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• CTX newborn screening is possible using only metabolite ratios. • Screening of 20,076 deidentified newborn blood spots showed no CTX screen positives. • CTX newborn screening is possible using either FIA-MS/MS or UPLC-MS/MS. Cerebrotendinous Xanthomatosis (CTX) is a treatable disorder of bile acid synthesis caused by deficiency of 27-sterol hydroxylase -encoded by CYP27A1 - leading to gastrointestinal and progressive neuropsychiatric symptoms. Biochemically, CTX is characterized by accumulation of the bile alcohol cholestanetetrol glucuronide (GlcA-tetrol) and the deficiency of tauro-chenodeoxycholic acid (t -CDCA) and tauro-trihydroxycholestanoic acid (t -THCA). To ascertain the feasibility of CTX newborn screening (NBS) we performed a study with deidentified Dutch dried blood spots using reagents and equipment that is frequently used in NBS laboratories. 20,076 deidentified newborn blood spots were subjected to flow-injection (FIA)-MS/MS and UPLC-MS/MS analysis to determine the concentration of GlcA-tetrol and calculate the GlcA-tetrol/ t -CDCA and t -THCA/GlcA-tetrol ratios. Using UPLC-MS/MS analysis both GlcA-tetrol concentration and/or metabolite ratios GlcA-tetrol/ t -CDCA proved to be informative biomarkers; newborn DBS results did not overlap with those of the CTX patients. For FIA-MS/MS, GlcA-tetrol also was an excellent marker but when using the combination of the GlcA-tetrol/ t -CDCA and t -THCA/GlcA-tetrol ratios also did not yield any screen positives. Newborn screening for CTX using only metabolite ratios following the measurement of three CTX biomarkers is possible using either FIA-MS/MS or UPLC-MS/MS, which paves the way for introduction of CTX NBS. [ABSTRACT FROM AUTHOR]

• An HPLC-DMS/MS/MS method was developed to assay steroid hormones in human serum. • A novel DMSPE method was developed to purify and to enrich the analytes in serum. • In situ derivatization was conducted on the adsorbent after DMSPE procedures. Androgens play important roles in polycystic ovarian syndrome (PCOS). However, measures of androgens based on mass spectrometry (MS) remain complex due to endogenous inferences of isomers or compounds with similar structures. Lack of sensitivity can also affect the accurate quantification of androgens, especially for very low level of 11-oxygenated androgens. We developed a fast and sensitive high-performance liquid chromatography-differential mobility spectrometry tandem mass spectrometry (HPLC-DMS/MS/MS) method for the simultaneous determination of seven androgens and 17-hydroxyprogesterone. Dispersive magnetic solid phase extraction (DMSPE) was conducted with core–shell structured nanoparticles of magnetic graphene oxide (Fe 3 O 4 @GO). In situ derivatization was performed using Girard's Reagent P. Linear ranges of the eight analytes were set in terms of clinical use. Intra- and inter-run precisions were < 16.7 % and 12.9 % for all the analytes and relative error was − 14.7–13.3 % and − 9.3–11.0 %, respectively. Extraction recoveries were 54.0–92.7 % for different analytes. The method was validated and was applied to assay 432 clinical samples. The developed method is green, fast, sensitive and accurate for the determination of endogenous androgens. It can be readily implemented in medical laboratories to provide superior analytical performance over the traditional electrochemiluminescence immunoassay method. [ABSTRACT FROM AUTHOR]

• The incidence of MADD was approximately 1:26,670. • Half of the MADD patients would not be detected under conventional screening protocols. • A novel MALDI-TOF MS assay targeting 23 ETFDH variants in the Chinese population was developed. • Incorporating second-tier genetic screening into the current NBS could improve the performance of MADD NBS. Newborn screening (NBS) for multiple acyl-CoA dehydrogenase deficiency (MADD) has poor sensitivity. This study aimed to evaluate the feasibility of incorporating second-tier genetic screening for MADD. A total of 453,390 newborns were screened for inherited metabolic disorders using tandem mass spectrometry from January 2017 to May 2022. A matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) assay was developed to identify 23 common ETFDH variants and used for second-tier screening of MADD. Overall, 185 newborns with suspected MADD received second-tier genetic screening. Thirty-three (17.8 %) newborns with positive results, of which 7 were homozygotes, 5 were compound heterozygotes, 21 were heterozygotes. Further genetic analysis revealed that 6 of the 21 newborns had a second ETFDH variant. Therefore, 18 patients were finally diagnosed with MADD, with a positive predictive value of 9.73 %. The detection rate and diagnostic rate of MALDI-TOF MS assay were 83.33 % and 66.67 %, respectively. Thus the incidence of MADD in our population was estimated at 1:25,188. Nine different ETFDH variants were identified in MADD patients. The most common ETFDH variant being c.250G > A with an allelic frequency of 47.22 %, followed by c.524G > A (13.89 %) and c.998A > G (13.89 %). All patients had elevation of multiple acylcarnitines at NBS. However, seven patients had normal acylcarnitine levels and two patients showed mild elevation of only two acylcarnitines during the recall review. We have established a high throughput MALDI-TOF MS assay for MADD screening. Half of the MADD patients would not be detected under conventional screening protocols. Incorporating second-tier genetic screening into the current NBS could improve the performance of MADD NBS. [ABSTRACT FROM AUTHOR]

• Cerebral creatine deficiency syndromes (CCDS) are metabolic disorders affecting creatine synthesis or transport. • Creatine and guanidinoacetate (GAA) are two biomarkers used to diagnose CCDS. • Different analytical methods were developed to measure the level of creatine and GAA in different body fluids. • In the current study a new UHPLC-MS/MS was developed. • Four novel intronic variants were discovered. Cerebral creatine deficiency syndromes (CCDS) are disorders affecting creatine synthesis or transport. Several methods have been developed to measure creatine and guanidinoacetate (GAA) in different body fluids including methods based on gas chromatography-mass spectrometry (GC–MS) and High-pressure liquid chromatography mass spectrometry (HPLC-MS). The diagnosis of CCDS is then confirmed by sequencing of creatine biosynthesis genes guanidinoacetate methyltransferase (GAMT) and Arginine: glycine amidinotransferase (GATM) and creatine transporter gene solute carrier family 6 member 8 (SLC6A8) or by functional enzymatic assay. The aim of the current study was to find the most reliable and accurate screening method for CCDS by comparing methods using Nuclear Magnetic Resonance spectroscopy (NMR), GC–MS and HPLC-MS. Additionally, this study was performed to estimate the prevalence of CCDS in a cohort of Egyptian patients and potentially to discover novel variants. The study was conducted on 150 subjects with clinical signs and symptoms consistent with CCDS. Metabolic profiling of urine samples was performed using three techniques: 1) GC–MS 2) Ultra high-pressure (or performance) liquid chromatography – Tandem Mass Spectrometry (UHPLC- MS/MS) and 3) NMR. The linearity of peak areas for creatine and GAA by UHPLC-MS/MS and NMR covered and exceeded the ranges normally found in urine. The limit of quantification and the inter-day precision results for creatine and GAA were more robust by UHPLC-MS/MS than NMR. Ten cases were identified as being positive for CCDS by our analytical approaches and underwent next generation sequencing (NGS) for GAMT, GATM and SLC6A8 genes. NGS was performed and confirmed one patient with one likely Pathogenic variant in GAMT gene: (NC_000019.10:g.1401317C > G, NP_000147.1:p.Ala54Pro). Additionally, we describe four novel intronic variants in the GATM gene: c.1043-357del and c.1043-357_1043-356insT, and were predicted to activate cryptic acceptor site with potential alteration of splicing, c.979-227G > A was found to significantly alter the Exon Splice Enhancer (ESE) xon Splice Silencer (ESS) motifs ratio and c.1042 + 262del which was found to have no implications on splicing. Both UHPLC-MS/MS and NMR spectroscopy are comparable to GC–MS in screening for CCDS. Nonetheless, the UHPLC-MS/MS method had better performance than NMR spectroscopy. Additionally, Sequencing of the full length of GATM, GAMT, and SLC6A8 genes is needed to identify intronic variants that could cause CCDS via affecting splice sites. [ABSTRACT FROM AUTHOR]

• Historical Perspective: Traces the evolution of inborn errors of metabolism (IEM) from Sir Archibald Garrod's initial hypothesis to modern advancements in molecular medicine. • Prevalence and Regional Variations: Discusses the estimated global prevalence of IEM, with a particular focus on the higher rates in the Eastern Mediterranean region due to consanguineous marriages. • Pathogenesis and Classification: Explores the genetic and biochemical basis of IEM, categorizing them into storage, intoxication, and energy deficiency disorders. • Diagnostic Modalities: Reviews various diagnostic approaches, from basic biochemical tests to advanced techniques like tandem mass spectrometry and next-generation sequencing. • Management Strategies: Evaluates therapeutic options, including dietary modifications, enzyme replacement therapy, pharmacological interventions, and emerging gene therapies. • Challenges in Pakistan: Highlights the specific challenges faced in diagnosing and managing IEM in Pakistan, such as high consanguinity rates and limited healthcare resources. • Future Directions: Suggests areas for further research, including the development of more effective screening programs, targeted therapies, and understanding the long-term outcomes of patients with IEM. There are many different genetic diseases called inborn errors of metabolism (IEM) which result from defective enzymes in the metabolic pathway. As a result, these defects either cause a harmful accumulation of substances or lead to a lack of certain types of molecule. The present review traces the origin and development of IEMs from Sir Archibald Garrod's theory in the early 20th century to current diagnostic and therapeutic approaches. It also involves a systematic literature review complying with PRISMA which included studies sourced from PubMed, Scopus, Web of Science and Google Scholar. It points out that high rates of consanguinity are associated with high prevalence rates for IEMs especially in the Eastern Mediterranean area. IEMS are classified as energy deficiency disorders, intoxication disorders, and storage disorders. Each category has a variety of clinical manifestations. This study incorporates different diagnostic methods ranging from simple biochemical tests to tandem mass spectrometry and next generation sequencing; while management approaches such as dietary modifications, enzyme replacement therapy and gene therapy were assessed for their efficacy. Specific attention is paid to Pakistan where there exists considerable consanguinity among people coupled with inadequate health care services which have seriously affected delivery of health care services thereby leading to numerous challenges for the country healthcare system during service provision. [ABSTRACT FROM AUTHOR]

• We overviewed the common pre-treatment workflows for protein detection using LC-MS/MS. • We outlined the superiority of LC-MS/MS in protein and peptide quantification compared to traditional immunoassays. • We reviewed the role of LC-MS/MS in qualitative analysis of proteins in clinical laboratories. • We summarized the challenges of LC-MS/MS in the measurement of peptides and proteins in clinical laboratories. Proteins are essential components of human cells and tissues, and they are commonly measured in clinical laboratories using immunoassays. However, these assays have certain limitations, such as non-specificity binding, insufficient selectivity, and interference of antibodies. More sensitive, accurate, and efficient technology is required to overcome these limitations. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is a powerful analytical tool that provides high sensitivity and specificity, making it superior to traditional methods such as biochemical methods and immunoassays. While LC-MS/MS has been increasingly used for detecting small molecular analytes and steroid hormones in clinical practice recently, its application for protein or peptide analysis is still in its early stages. Established methods for quantifying proteins and peptides by LC-MS/MS are mainly focused on scientific research, and only a few proteins and peptides can be or have the potential to be detected and applied in clinical practice. Therefore, this article aims to review the clinical applications, advantages, and challenges of analyzing proteins and peptides using LC-MS/MS in clinical laboratories. [ABSTRACT FROM AUTHOR]

• The incidence of HPA in Quanzhou, China was estimated to be 1 in 10,365 newborns. • The incidences of PAHD and BH 4 D in Quanzhou were 1:12,350 and 1:64,496, respectively. • The incidence of BH 4 D was as high as 15.1% in the southern Chinese population. • The most common PAH variants were c.158G > A (22.3 %) and c.721C > T (14.9 %). • The most common PTS variants were c.155A > G (50.0 %) and c.259C > T (33.3 %). Hyperphenylalaninemia (HPA) is the most common congenital amino acid metabolism-related defect, but its incidence differs substantially between northern and southern China. We aimed to elucidate the incidence, proportion, and genetic features of HPA in a southern Chinese population. We analyzed the HPA screening results for 580,460 newborns from 2014 to 2021. Of the 296 newborns who tested HPA positive, 56 were diagnosed with HPA, including 47 with phenylalanine hydroxylase deficiency and nine with tetrahydrobiopterin deficiency (BH 4 D). HPA incidence was estimated to be 1:10,365 newborns. All patients had elevated Phe and Phe/Tyr levels. Thirty-three PAH variants and five PTS variants were detected in HPA patients; 80.6 % PAH variants and 100 % PTS variants were classified as pathogenic or likely pathogenic. In silico tools predicted the remaining variants to be damaging. PAH variants clustered in exons 3, 5, 7, 11, and 12 and PTS variants clustered in exons 2 and 5. The most common PAH variants were c.158G > A (p.R53H, 22.3 %) and c.721C > T (p.R241C, 14.9 %). The most common PTS variants were c.155A > G (p.N52S, 50.0 %) and c.259C > T (p.P87S, 33.3 %). Newborn screening is an effective method for early detection of HPA, but differential diagnosis of BH 4 D is necessary. [ABSTRACT FROM AUTHOR]

[Display omitted] • Simultaneous quantitation of 23 types and sites of modification in human hemoglobin. • Tyrosine chlorination and nitrosylation are higher in gastric cancer patients. • Conversion of histidine to aspartate is lower in gastric cancer patients. • Advanced oxidation of histidine to hydroxyaspartate is higher in gastric cancer patients. Overproduction of the reactive oxygen, nitrogen, and chlorine species by the immune systems during chronic infection and inflammation can cause structural and functional changes of cellular proteins. The high abundance of hemoglobin in blood makes hemoglobin adducts suitable biomarkers for assessing the damage of these reactive species in the body. In this study, a total of 23 types and sites of modification in human hemoglobin were simultaneously analyzed, including monooxygenation of histidine, tyrosine, methionine, and aspartate, conversion of histidine to aspartate and hydroxyaspartate, as well as chlorination and nitration of tyrosine residues. Hemoglobin was isolated from the blood of the study subjects, digested into peptides, and the extents of these modifications were quantified relative to their parent peptides using nanoflow liquid chromatography nanoelectrospray ionization tandem mass spectrometry under selected reaction monitoring (nanoLC − NSI-MS/MS-SRM). The extents of monooxygenation at β-His-77 and β-Tyr-130, chlorination at α-Tyr-24 and β-Tyr-130, and nitrosylation at α-Tyr-24 were elevated in gastric cancer patients. Conversely, conversion of histidine to aspartate at α-His-20, α-His-50, β-His-2, β-His-143, and monooxygenation at β-His-143 were decreased in gastric cancer patients. The areas under the receiver operating characteristics (AUC of ROC) curve of these ten types and sites of hemoglobin modifications were between 0.7644 and 0.9644. The ratio of conversion of histidine to hydroxyaspartate versus conversion of histidine to aspartate was significantly higher in gastric cancer patients at α-His-20, α-His-50, and β-His-143 (p < 0.05) with AUC of ROC ranging between 0.7689 and 0.9178. To our knowledge, this is the first report of simultaneous measurement of multiple types of oxidative and advanced oxidative hemoglobin modifications in gastric cancer patients. The results revealed elevated levels of oxidative stress-induced protein damage in gastric cancer patients and the potential of using these modifications of hemoglobin as biomarkers for evaluation of oxidative stress in one drop of blood. [ABSTRACT FROM AUTHOR]

• Current immunoassays for opioid detection lack specificity. • DART-MS/MS has the potential to fill the gap between immunoassays and LC-MS/MS. • DART-MS/MS is validated using clinical samples for opioid detection in urine. • DART-MS/MS showed high sensitivity and specificity for detection of most analytes. • Ion ratios can help distinguish between isobaric compound pairs. Current laboratory methods for opioid detection involve an initial screening with immunoassays which offers efficient but non-specific results and a subsequent liquid chromatography-tandem mass spectrometry (LC-MS/MS) confirmation which offers accurate results but requires extensive sample preparation and turnaround time. Direct Analysis in Real Time (DART) tandem mass spectrometry is evaluated as an alternative approach for accurate opioid detection with efficient sample preparation and turnaround time. DART-MS/MS was optimized by testing the method with varying temperatures, operation modes, extraction methods, hydrolysis times, and vortex times. The method was evaluated for 12 opioids by testing the analytical measurement range, percent carryover, precision studies, stability, and method-to-method comparison with LC-MS/MS. DART-MS/MS shows high sensitivity and specificity for the detection of 6-acetylmorphine, codeine, hydromorphone, oxymorphone, hydrocodone, naloxone, buprenorphine, norfentanyl, and fentanyl in urine samples. However, its performance was suboptimal for norbuprenorphine, morphine and oxycodone. In this proof-of-concept study, DART-MS/MS is evaluated for its rapid quantitative definitive testing of opioids drugs in urine. Further research is needed to expand its application to other areas of drug testing. [ABSTRACT FROM AUTHOR]

• "Tranq-dope", xylazine-adulterated opioids, has caused many overdose deaths in the US. • Xylazine and opioids act in synergy, increasing the lethality of one another. • We report the first fatal case of xylazine-adulterated heroin in the EU. • Xylazine and its main metabolite were detected in blood and urine by LC-HRMS/MS. • Xylazine should be strictly controlled by public health authorities and toxicologists. The number of xylazine-involved overdose deaths tremendously increased from 2019 onwards in the US. This is due to the "tranq-dope" trend consisting in mixing opioids with the sedative to reduce drug manufacturing costs and enhance their effects. In this study, we report the first fatality involving xylazine-adulterated heroin in the EU. The subject was a 33-year-old Caucasian male with a documented history of drug abuse who was found dead in a public area with puncture marks at the elbow. Peripheral blood and urine were collected at the autopsy and analyzed by liquid chromatography-high-resolution tandem mass spectrometry (LC-HRMS/MS) after protein precipitation. 6-Monoacetylmorphine, total/free morphine, and codeine blood concentrations of 20.3, 236/105, and 38.3 ng/mL, respectively, indicated recent heroin consumption. Methadone blood concentration was below 10 ng/mL. Alprazolam, nordiazepam, and flurazepam blood concentrations were 23.9, 61.4, and 55.0 ng/mL, respectively. Benzoylecgonine blood concentration was below 5 ng/mL. Xylazine blood and urine concentrations were 105 and 72.6 ng/mL, respectively. The combination of central nervous system depressants, i.e., opioids, benzodiazepines, and xylazine, was the principal cause of death by cardiorespiratory failure. The case was promptly reported to the UE Early Warning System on drugs. [ABSTRACT FROM AUTHOR]

• Protonitazene is a novel synthetic opioid, which belongs to the nitazene family. • Nitazenes have re-emerged on the new psychoactive substances market and have been reported in several fatal intoxication cases. • Protonitazene metabolism was studied in vitro after drug incubation with human liver microsomes. • The identified metabolites were compared to the responses obtained in two authentic human urine specimens.. Protonitazene, or N,N-diethyl-5-nitro-2-[(4-propoxyphenyl)methyl]-1H-benzimidazole-1-ethanamine, is a novel synthetic opioid, which belongs to the nitazene family. Over the last four years, nitazenes have re-emerged on the new psychoactive substances market and have been reported in several fatal intoxication cases. The metabolism of several nitazene analogues have already been studied, but to date, no data exists regarding protonitazene. The aim of the study was the detection of protonitazene and its metabolites in authentic human urine collected in two fatal intoxication cases, comparing the data after in vitro incubation with human liver microsomes, and subsequent analysis by ultra-performance liquid chromatography-tandem mass spectrometry and ultra-performance liquid chromatography-high-resolution mass spectrometry. Protonitazene metabolites, including N-desethyl-protonitazene, 5-amino-protonitazene and 4-hydroxy-nitazene, were characterized in vitro and were identified in the urine of both cases. The ratios between metabolites and parent protonitazene, higher than 1, were calculated to estimate the proportionality of metabolites. The results suggest that testing protonitazene metabolites should increase the window detection of exposure to protonitazene. [ABSTRACT FROM AUTHOR]

• Tandem mass spectrometry enables rapid and accurate detection of metabolites. • Tandem mass spectrometry for neonates and high-risk infants can provide a wealth of clinical data and metabolite spectra. • Early detection and early diagnosis can lead to more precise treatment, genetic counseling and scientific research. Compare the differences between normal newborns and high-risk children with inherited metabolic diseases. The disease profile includes amino acidemias, fatty acid oxidation disorders, and organic acidemias. Data was collected on newborns and children from high-risk populations in Shanghai from December 2010 to December 2020. 232,561 newborns were screened for disorders of organic, amino acid, and fatty acid metabolism. The initial positive rate was 0.66 % (1,526/232,561) and the positive recall rate was 77.85 %. The positive predictive value is 4.71 %. Among them, 56 cases were diagnosed as metabolic abnormalities. The total incidence rate is 1:4153. Hyperphenylalaninemia and short-chain acyl-CoA dehydrogenase are the most common diseases in newborns. In addition, in 56 children, 39 (69.42 %) were diagnosed by genetic sequencing. Some hotspot mutations in 14 IEMs have been observed, including PAH gene c.728G > A, c.611A > G, and ACADS gene c. 1031A > G, c.164C > T. A total of 49,860 symptomatic patients were screened, of which 185 were diagnosed with IEM, with a detection rate of 0.37 %. The most commonly diagnosed diseases in high-risk infants aremethylmalonic acidemia and hyperphenylalaninemia. There are more clinical cases of congenital metabolic errors diagnosed by tandem mass spectrometry than newborn screening. The spectrum of diseases, prevalence, and genetic characteristics of normal newborns and high-risk children are quite different. [ABSTRACT FROM AUTHOR]

• Gestational vitamin D supplementation should be continued also during summer months. • 3-epi-25-hydroxyvitamin D3 was detectable in all cord blood specimens. • C3-epimerization mainly depends on cord blood 25-hydroxyvitamin D3 concentration. High prevalence of hypovitaminosis D is worldwide reported among pregnant women and newborns. We assessed cord blood 25-hydroxyvitamin D 3 [25(OH)D 3 ] and 3-epi-25-hydroxyvitamin D 3 (C 3 -epimer) levels in relation to assumed maternal risk factors for hypovitaminosis D. We enrolled 246 term newborns during summer. 175/246 mothers were supplemented with a daily variable dosage (200–1,000 IU) of vitamin D 3 during pregnancy. Cord blood 25(OH)D 3 and C 3 -epimer concentrations were analyzed by high performance liquid chromatography tandem mass spectrometry. Median cord blood 25(OH)D 3 levels were 23.4 ng/mL (16.9–28.8). The prevalences of vitamin D sufficiency (≥ 30.0 ng/mL), insufficiency (20.0–29.9 ng/mL), and deficiency (< 20.0 ng/mL) were 19.9%, 45.9%, and 34.2%, respectively. Non-Caucasian ethnicity, housewife life, weight excess, negligible sun exposure and absent gestational vitamin D supplementation were associated with both reduced cord blood 25(OH)D 3 and C 3 -epimer levels. C 3 -epimer/25(OH)D 3 ratio was 15.1% (13.6%-18.4%) and it was not related to any of the assumed risk factors for hypovitaminosis D. Cord blood vitamin D deficiency was common, particularly in newborns from mother not receiving vitamin D supplementation and with poor sun exposure. C 3 -epimer levels were high in cord blood, causing possible misclassification of vitamin D status if they were not distinguished from 25(OH)D 3 concentrations. [ABSTRACT FROM AUTHOR]

• Revealed the region-wise difference of 8 hair steroid hormone concentrations. • Some androgens have the correlation with AGA's clinical presentation. • Dihydrotestosterone in hair follicle tissue reflected the local hormone synthesis. • Hair testosterone and cortisol levels were contributed from peripheral blood. Steroid hormones are known to be associated with diseases like androgenetic alopecia (AGA) resulting in hair loss. The lack of a detailed analysis of the local concentration of steroids in different parts of the head underlies the rationale and purpose of this study. To evaluate the concentration distributions of steroid hormones in hair in different parts of the head, hair samples of 8 healthy men from 9 point-areas covering the frontal, parietal, and occipital regions were collected. Eight steroid hormones were measured by using the LC-MS/MS and region-wise comparison for different hormones was done using the mean z-score and Tukey's HSD. Five of the 8 hormones had a high concentration in the parietal region, with dihydrotestosterone (DHT) showing a peak in the central parietal region (z = 1.59) suggesting a correlation with AGA's clinical presentation. Whereas, no significant differences were observed for testosterone and cortisol between the parietal and occipital regions. Higher DHT levels at the parietal region were also verified with a small group of AGA patients. This research expands upon the role of steroid hormones in hair follicle tissue elucidating their relationship with disease, thus contributing to disease management. [ABSTRACT FROM AUTHOR]

• β-chain glycation in HbG-Coushatta and HbG-Taipei was determined using LC-MS/MS. • Glycation rates of βG-Coushatta and βG-Taipei were significantly higher than βA. • Overall glycation of HbAG-Coushatta and HbAG-Taipei was higher than HbAA. Hemoglobin A 1c (HbA 1c) is widely used to measure glycemic status and control in diabetes testing and treatment and is an important risk factor forcomplications of diabetes. Hemoglobin variants can interfere with the HbA 1c testing method by reducing the life span of erythrocytes or due to differences in glycation degrees. In this study, glycation levels of the HbA, HbG-Coushatta, and HbG-Taipei β-chains (βA, βG-Coushatta, and βG-Taipei, respectively) were examined. Blood samples from heterozygotic patients (HbG-Coushatta/HbA, HbG-Taipei/HbA) were analyzed. Glycation rateswere determined using high-performance liquid chromatography with tandem mass spectrometry. Ratios of glycated βG-Coushatta to glycated βA and glycated βG-Taipei to glycated βA were calculated by comparingareas under the curves from extracted ion chromatograms. βG-Coushatta and βG-Taipei were 6.08 ± 1.38% and 5.95 ± 0.93% glycated (respectively), which were significantly higher than βA chains(4.55 ± 1.30% and 4.51 ± 0.91%, respectively; p = 0.000). The total glycation degree (α + β) in HbG-Coushatta and HbG-Taipei heterozygotes were estimated to be 9% and 8% higher than those of HbA homozygotes (P＜0.001), respectively. βG-Coushatta and βG-Taipei glycation degrees were significantly higher than βA, while the differences in total hemoglobin (α + β) were small and unlikely to impact the clinical interpretation of HbA 1c results. [ABSTRACT FROM AUTHOR]

[Display omitted] • New biomarkers prognostic of proximal urea cycle disorders are strongly desirable. • Metabolic profiles of affected patients show high value of glutamine to glutamate ratio. • The ratio can be considered a more effective marker than citrulline and glutamine. • The ratio can be included in a newborn screening panel. Proximal urea cycle disorders (PUCDs) are not included in most newborn screening programs due to the lack of adequate markers to monitor. Failure to alter citrulline and glutamine levels, the prognostic markers commonly used, can results in high false negative. Therefore, new biomarkers, prognostic of PUCDs, are strongly desirable. We used tandem mass spectrometry to analyze blood spot from PUCDs patients during their follow up in our referral center focusing on glutamine to glutamate (Gln/Glu) ratio. We reanalyzed the same specimens of three patients after two months and the specimen of a new patient with suspicious of PUCD disorder. Specimens of our patients shown a significant elevation of the ratio Gln/Glu compared to that of a healthy population (p < 0.05) as well as the specimens analyzed after two months, while the glutamine concentration dropped. New patient, showing high value of the ratio, was molecularly confirmed as PUCD patient. We further analyzed the blood spots from a neonatal population in order to fix a cut-off value and include it in a newborn screening panel. Our preliminary results suggest that the Gln/Glu ratio could be a very useful diagnostic marker, more stable over time than glutamine, which could improve the performance in early PUCDs identification. [ABSTRACT FROM AUTHOR]

• A specific LC-MS/MS method to measure seven steroids simultaneously from DBS to diagnose CAH. • DHEA and pregnenolone do not require derivatization during sample preparation. • The data demonstrates a functional framework for steroid analysis in 6 mm DBS samples by LC-MS/MS. • Baseline separation of six steroids was successfully achieved under 7.5 min. • This method is validated according to CLSI guidelines. Congenital adrenal hyperplasia (CAH) is a group of autosomal recessive disorders that occur due to defects in the steroidogenesis pathway. Approximately 90% of CAH cases can be diagnosed by the measurement of serum 17-hydroxyprogesterone alone. However, the quantification of six additional steroids could significantly improve CAH laboratory diagnosis. Using dried blood spot (DBS) as specimen of choice can further improve patient care due to the small sample volume required for CAH diagnosis in neonates. An optimized DBS sample preparation method was employed for steroids quantification without the need of derivatization. A LC-MS/MS assay was developed and optimized using a reverse phase-ultra high-pressure liquid chromatography (RP-UHPLC) system combined with triple quadrupole mass spectrometry using positive electrospray ionization mode. The assay was validated according to CLSI analytical guidelines, including lower limit of quantification (LLOQ), linearity, precision, accuracy, carryover, and method comparison. The analytical measuring range of the method for all steroids was 2.5, 5, or 10 ng/ml to 250 ng/ml in DBS, r2 ≥ 0.995. The LLOQ, intra-day and inter-day precision were 0.11–1.8 ng/ml, 1.2–6.4 ng/ml, 1.8–11.5%, and 5.3–13.8%, respectively. Our LC-MS/MS assay simultaneously detects 7 steroids for the diagnosis of CAH and can be readily implemented in clinical laboratories to provide superior analytical performance over traditional immunoassays. [ABSTRACT FROM AUTHOR]

• Measurement of residual GALC activity in leukocytes to diagnose Krabbe disease. • Pre-analytical and analytical variability were evaluated and clinically validated. • LC-MS/MS confirmatory assay for multiple lysosomal enzymes was developed. Deficiency of galactosylcerebrosidase (GALC) causes Krabbe disease. Historically, a diagnosis is made by measuring GALC enzymatic activity with a radioisotope assay. To improve the workflow and performance, we developed and clinically validated a leukocyte enzymatic assay using liquid chromatography tandem mass spectrometry (LC-MS/MS). Extracted cell lysates were quantified and incubated with commercially available multiplexed substrates and internal standards. Liquid-liquid extraction was performed, and pre-analytical and analytical variability were evaluated and validated following clinical laboratory regulation guidelines. Enzymatic reaction products were resolved from substrate breakdown products by a 3.5-minute column separation. Intra- and inter- assay imprecision were less than 15%. No matrix effects or carryover were observed. ACD anticoagulant tubes provide the best sample stability. Detection of product was linear with an R2 of 0.99. Small differences in GALC activity were measurable near the anticipated disease range. Confirmed cases of Krabbe disease were well differentiated from carriers and non-Krabbe individuals (normal reference range). An LC-MS/MS assay was developed, which can measure trace residual GALC activity in leukocytes and aid in the diagnosis of Krabbe disease. The multiplexed mixture allows for built-in sample quality control and enables a streamlined workflow for evaluation of multiple lysosomal storage diseases. [ABSTRACT FROM AUTHOR]

• Development of a candidate reference method for serum creatinine determination correcting creatine-creatinine interconversion. • Two-dimensional liquid chromatography coupling reversed phase and cation exchange separation minimises matrix effects. • Quantification is carried out by multiple linear regression and double spiking isotope dilution tandem mass spectrometry. • Creatine-creatinine interconversion during sample preparation was evaluated with the analysis of 93 serum samples. • Negligible creatine-creatinine interconversion was found in all the analysed samples using the proposed sample preparation. Development of a candidate reference method based on bidimensional liquid chromatography coupled to ESI-MS/MS and double spike isotope dilution for serum creatinine quantification capable of correcting for creatinine-creatine interconversion during sample pretreatment. Study of the impact of the creatine-creatinine interconversion during the analysis of human serum samples. 13C 1 -creatinine and 13C 2 -creatine are added to the serum sample. Separation carried out by bidimensional liquid chromatography combining reversed phase and a strong cation exchange chromatography. The heart cut, containing creatine and creatinine, is automatically transferred to the second dimension. Quantification carried out by double spike isotope dilution tandem MS/MS. Minimization of spectral interferences and ion suppression due to matrix effects while increasing sample throughput compared to the direct coupling of cation exchange chromatography to the ESI source. Trueness of the method studied with the satisfactory analysis of two certified reference materials. Satisfactory intra- and inter-day precisions obtained analysing a serum pool and control sera. Analysis of 93 serum samples revealed negligible interconversions with no correlation with creatine levels. The method provides adequate analytical figures of merit for serum creatinine determination according to CSLI guidelines. Negligible creatine-creatinine interconversion is promoted with the applied sample preparation procedure. [ABSTRACT FROM AUTHOR]

• Saliva, sweat, hair, and meconium are emerging types of specimen for drug testing. • LC-MSMS becomes a new gold standard for confirmatory drug testing. • LC-hybrid-Tof-MS is usable for comprehensive drug screening. • Miniature ambient ionization MS can quickly analyze biological specimens. • Miniature ambient ionization MS is promising for rapid drugs of abuse testing. Drugs of abuse testing is widely used clinically and forensically. Urine is the preferred type of specimen for drugs of abuse screening, but saliva, sweat, hair, and meconium are emerging types of specimens. GC–MS has been used as a gold standard for confirmatory drug testing, but LC-tandem-MS can analyze more diverse types of analytes than GC–MS. Thus, LC-tandem-MS becomes a new gold standard for confirmatory drug testing. Unlike GC–MS, LC-tandem-MS is not suited for non-targeted comprehensive drug screening. But with the advent of high-resolution-MS such as Tof-MS, which can discriminate the compounds of similar molecular masses but with different formulas, LC-hybrid-Tof-MS is usable for non-targeted comprehensive drug screening. Another technical advancement is the advent of miniature ambient ionization MS, which can analyze biological specimens including urine within one minute. Thus these mass spectrometers are promising for rapid drugs of abuse testing in a POC setting. [ABSTRACT FROM AUTHOR]

• The incidence of PCD in children in the Quanzhou area was 1:10126. • Eighteen SLC22A5 variants were found, with five novel ones. • The most prevalent variant in neonatal and maternal patients was c.760C > T (p.R254*) • Newborn screening is effective in identification and timely treatment of PCD. Primary carnitine deficiency (PCD) is an autosomal recessive disease caused by functional defects in the carnitine transporter OCTN2 due to mutations in SLC22A5. Here, we aimed to understand the incidence, clinical, biochemical, and molecular features of PCD in Quanzhou, China. Newborn screening (NBS) was performed through tandem mass spectrometry (MS/MS) to detect genetic metabolic diseases. Next-generation sequencing was used to detect SLC22A5 mutations in patients with suspected PCD. From 364,545 newborns screened, 36 were diagnosed with PCD, in addition to five mothers. The incidence of PCD in children in the Quanzhou area was 1:10126. Eighteen SLC22A5 variants were found, with five novel ones. The most prevalent variant in neonatal and maternal patients was c.760C > T (p.R254*). Twenty-five neonatal patients received L-carnitine supplementation; however, one patient discontinued treatment and sudden death occurred. One sibling presented repeated fatigue, hypoglycemia, and coma, but the symptoms disappeared after treatment. Two mothers with PCD claimed to feel weak and easily fatigued. The incidence of PCD is relatively high in the Quanzhou area. Five novel variants were found, broadening the mutation spectrum of SLC22A5. NBS is effective in identifying PCD, and sudden death may be prevented with timely treatment. [ABSTRACT FROM AUTHOR]

• Intact glucagon can be simply and accurately measured by LC-MS/MS. • Fast and easy sample preparation with solid-phase extraction is suitable to routine application. • The described method can open up the possibility to standardize glucagon analysis. The quantitation of glucagon remains a challenging immunoassay, mainly due to cross-reactivity. A sensitive, rapid and specific intact glucagon method is therefore necessary for quality routine analysis. A tandem mass spectrometry method to fulfill this objective is described in this work. Glucagon was extracted from plasma employing a mixed-mode anion exchange solid-phase extraction. Sample stability was assessed in K2-EDTA and P800 tubes at different temperatures. We compared our method to two different immunoassays. FDA and EMA guidelines were followed for validation. An external quality control program served for comparison with other laboratories. Assay imprecision was below 4%. Recoveries were within 95–103%. LoQ was 8.75 pg/mL. Total analytical CV was 2.91%. Samples were found stable at 4 °C for less than 4 h. Diasource® RIA disagreed with our method. Mercodia® ELISA provided a closer agreement, also proven by external quality control samples. A rapid and specific LC-MS/MS method for glucagon quantitation has been developed, validated and is suitable to routine care. The simplicity and the good performances in terms of time and specificity, could open the possibility to establish a standardized method for glucagon. [ABSTRACT FROM AUTHOR]

• HPLC–MS/MS method for the quantification of nine diverse antibiotics. • Includes trimethoprim-sulfamethoxazole, ciprofloxacin, ertapenem and five cephalosporins. • Unbound ertapenem and cefazolin can be measured after ultracentrifugation. • Extensive stability data are presented. • The method was implemented in routine TDM and in a clinical study. TDM of antibiotics can bring benefits to patients and healthcare systems by providing better treatment and saving healthcare resources. We aimed to develop a multi-analyte method for several diverse antibiotics using LC-MS/MS. Sample preparation consisted of protein precipitation with methanol, dilution and online extraction using a Turboflow Cyclone column. Separation was performed on a Synergi 4 µm Max RP column and deuterated forms of three antibiotics were used as internal standards. We present a LC-MS/MS method for the quantitative determination of nine antibiotics, including five cephalosporins, the carbapenem ertapenem, the fluoroquinolone ciprofloxacin as well as the combination drug trimethoprim-sulfamethoxazole from plasma. Additionally, unbound ertapenem and cefazolin were analyzed in plasma water after ultrafiltration using plasma calibrators. Results from routine TDM show the applicability of the method. The presented method is precise and accurate and was introduced in a university hospital, permitting fast TDM of all nine analytes. It was also used in a clinical study for measuring cefazolin free and total concentrations. [ABSTRACT FROM AUTHOR]

• The study comprehensively analyzed the newborn screening data of metabolism disorder of 189,354 infants in Ningxia Hui Autonomous Region of China and demonstrated the ethnic preference of the disease incidence and genetic variant distribution. • The study provides insights on multi-ethnic patients in China. Chinese newborns have been screened for inborn errors of metabolism (IEM) for over 20 years. Although China features 56 different ethnic groups, there are no specific data describing the incidence of such genetic errors across difference ethnicities. To understand the ethnic preference distribution of the incidence and variants of IEM in the Ningxia Hui Autonomous Region of China, 189,354 newborns from 2016 to 2019 were screened by tandem mass spectrometry, including 87,482 from the Han ethnic population, 88,229 from the Hui population, 1,103 from other ethnicities, and 12,540 infants without ethnic registration. Suspected cases then underwent specific genetic profiling by targeted next generation sequencing. In total, 160 patients were diagnosed with 17 types of IEM, with a significant higher incidence in Hui infants (1/1,003) than in Han infants (1/1,232). Five diseases (eight patients) were specifically detected in Han infants, while three were exclusively diagnosed in six Hui infants. For shared diseases, the variants of keys genes also showed ethnic preference. Our findings enhance our understanding of the genetics underlying IEM, thus promoting the development of treatment plans for patients from different areas or ethnicities in China. [ABSTRACT FROM AUTHOR]

• Pre-symptomatic citrulline concentrations on NBS were significantly low in late-onset OTCD patients. • Hypocitrullinemia on NBS can be used as a screening marker for some patients with late-onset OTCD. • Further retrospective and prospective study of pre-symptomatic citrulline concentrations should be conducted. Ornithine transcarbamylase deficiency (OTCD) is the most common urea cycle disorder. Late-onset OTCD manifests after the neonatal period; therefore, if pre-symptomatic diagnosis and treatment are performed, it can improve the prognosis by preventing hyperammonemia. However, pre-symptomatic diagnosis is unreliable as the specific screening marker of OTCD has not been established yet. This retrospective study aimed to evaluate the pre-symptomatic blood citrulline levels in patients with late-onset OTCD. Patients with late-onset OTCD who were born after the newborn screening based on tandem mass spectrometry (MS/MS-NBS) was started and were referred to Hyogo College of Medicine Hospital between 2014 and 2018 were included. Pre-symptomatic blood citrulline levels measured by MS/MS-NBS were retrospectively evaluated. Four patients were included in this study. The pre-symptomatic blood citrulline levels were 2.02, 4.50, 4.97, and 3.75 µmol/l, respectively. Compared with the citrulline levels in all newborns in Hyogo prefecture, these values were significantly low. These results suggest the possibility that hypocitrullinemia detected by the MS/MS-NBS can be used as a screening marker for some patients with late-onset OTCD. Further retrospective evaluation of pre-symptomatic citrulline levels in patients with late-onset OTCD, as well as prospective monitoring of hypocitrullinemia on the MS/MS-NBS should be conducted. [ABSTRACT FROM AUTHOR]

• Compilation of the recent literature on clinical samples. • Presentation of the accurate methodology. • Sample preparation. • Information on separation and identification of neutral, hydrophobic and hydrophilic lipids. • Compilation of current statistical methods used in mass spectrometry of glycero-, glycerophospho- and sphingolipids. The present article examines recently published literature on lipids, mainly focusing on research involving glycero-, glycerophospho- and sphingo-lipids. The primary aim is identification of distinct profiles in biologic lipidomic systems by ultra-high-performance liquid chromatography (UHPLC) coupled with mass spectrometry (MS, tandem MS) with multivariate data analysis. This review specifically targets lipid biomarkers and disease pathway mechanisms in humans and artificial targets. Different specimen matrices such as primary blood derivatives (plasma, serum, erythrocytes, and blood platelets), faecal matter, urine, as well as biologic tissues (liver, lung and kidney) are highlighted. [ABSTRACT FROM AUTHOR]

To validate a liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for the measurement of glycosaminoglycans (GAGs) in plasma and serum. To establish plasma, cerebrospinal fluid (CSF) and urine reference intervals. To compare GAGs in serum with that in urine and CSF from patients with MPS I. Dermatan sulfate (DS), heparan sulfate (HS), and chondroitin sulfate (CS) in serum/plasma, urine and CSF were methanolysed into dimers and analyzed using pseudo isotope dilution UPLC-MS/MS assay. Serum, CSF and urine DS and HS were quantified for 11 patients with mucopolysaccharidosis (MPS) type I before and after treatment with Aldurazyme® (laronidase) enzyme replacement therapy (ERT). The method showed acceptable imprecision and recovery for the quantification of serum/plasma CS, DS, and HS. The serum, urine, and CSF DS and HS concentrations were reduced after 26 weeks of ERT in 4 previously untreated patients. Serum DS and HS concentrations normalized in some patients, and were mildly elevated in others after ERT. In contrast, urine and CSF DS and HS values remained elevated above the reference ranges. Compared with serum GAGs, urine and CSF DS and HS were more sensitive biomarkers for monitoring the ERT treatment of patients with MPS I. [ABSTRACT FROM AUTHOR]

N-acetylaspartate is a human endogenous compound synthesized by neurons, which is involved in neuronal metabolism. It is used as a marker in brain magnetic resonance spectroscopy to investigate several neurological and metabolic disorders, that can be related to a variation of its concentration with respect to reference values. N-acetylaspartate is present also in biological fluids, such as plasma, urine, and cerebrospinal fluid, where it can be quantified. Here we describe the development and validation, in compliance with the EMA guidelines, of a novel assay method for the quantification of N-acetylaspartate in plasma based on tandem mass spectrometry coupled to liquid chromatography. Its peculiarity lies in the fact that sample preparation includes an esterification step, which significantly improves the chromatographic performances and, consequently, also the method sensitivity, reproducibility and accuracy. Instrumental LLOQ is 0.06 ng/mL, i.e. at least 300 times lower than the medium N-acetylaspartate concentration in samples, accuracy is in the range 98–103%, while precision lies between 1 and 3%. The method robustness was tested in about 1000 injections of plasma samples, 96 of which were used also to assess the reference ranges in control subjects (16.46–63.40 ng/mL). Controls were then compared to plasma samples from type 2 diabetic patients. Contrary to brain magnetic resonance spectroscopy, which demonstrated a decrease in the N-acetylaspartate levels in right frontal and parieto-temporal region of type 2 diabetic patients, plasma analysis showed no statistical difference with respect to controls. However, the method here described can be profitably used in studies concerning different disorders with CNS involvement, as confirmed by reports available in the literature. [ABSTRACT FROM AUTHOR]

• LC-MS/MS and chemiluminescent immunoassay results of four immunosuppressive drugs in patient samples were compared. • Cyclosporine A concentration in blood from liver transplantation patients was found assay dependent. • Sedimentation of blood cells effects the measured concentration. • Hemolyzed whole blood is an alternative sample form. Life-long monitoring of immunosuppressive drugs (ISDs) in blood is essential after organ transplantation. However, the ISD concentrations vary depending on the assay employed. ISDs are strongly bound to cytoplasmic proteins in erythrocytes in circulation. Therefore, the relatively rapid sedimentation of blood cells in whole blood samples may affect the results when using liquid handling robots. We used 1115 blood samples from outpatients and ward patients with kidney (n = 373), liver (n = 101), heart (n = 29) and bone marrow (n = 155) transplant. Whole blood samples were pretreated by protein precipitation. Alternatively, the samples were hemolyzed by freezing prior to precipitation. ISDs were analyzed by a 2-plexing liquid chromatography tandem mass spectrometry (LC-MS/MS) assay and commercial chemiluminescent microparticle immunoassays (CMIA). The difference between the two sample preparation practices was negligible (<2%). Overall, the measured ISD concentrations in patient samples were lower by LC-MS/MS than by CMIA. The difference was the largest (20.2%) and the smallest (9.1%) in samples from liver and from heart transplant patients, respectively. CMIA overestimates blood ISD concentrations as compared to LC-MS/MS. The extent of the difference was found to be organ transplant dependent. The ISDs can be quantitated either from intact or hemolyzed blood samples. [ABSTRACT FROM AUTHOR]

• This study reported the largest series of patients with PCD detected by NBS. • The incidence of newborns with PCD in Zhejiang province was 1:30,182. • Thirty-seven distinct variants were identified in SLC22A5 , of which 10 were novel. • c.1400C>G (p.S467C) was the most common variant in newborns and mothers with PCD. • c.760C>T (p.R254*) was rarely detected in the maternal PCD patients. Testing for primary carnitine deficiency (PCD) has been implemented in many newborn screening (NBS) programs, but few large-scale studies on NBS for PCD have been reported in China. This study aimed to assess the incidence and biochemical, clinical, and genetic characteristics of PCD discovered by NBS. Dried blood spots from newborns were analyzed by tandem mass spectrometry (MS/MS) and suspected positive patients were further tested using molecular genetic analysis. Infants who carried two variants in SLC22A5 or those with extremely low free carnitine levels during recall were referred for follow-up and treatment. Over 3.4 million newborns were screened and 113 newborns were diagnosed with PCD, yielding a positive predictive value of 1.93%. In addition, 63 mothers with PCD were identified. The incidence of PCD in newborns and mothers in Zhejiang was 1:30,182 and 1:54,137, respectively. Thirty-seven distinct variants were identified in SLC22A5 of which 10 were novel. c.1400C > G (p.S467C) was the most prevalent variant in both newborns and mothers with PCD, while c.760C > T (p.R254*), which is reportedly common in other Chinese regions, was rarely detected in maternal PCD patients. This study reports the largest series of patients with PCD detected by NBS and identifies 10 novel variants, expanding the variant spectrum of SLC22A5. [ABSTRACT FROM AUTHOR]

• NUS1-CDG can be detected by analysis of dolichol isoforms. • Ratio of dolichol isoforms changes during ageing. • New diagnostic method for CDG patients via tandem mass spectrometry. Dolichol is a membrane lipid which carries monosaccharides and glycans for N -linked protein glycosylation occurring in the endoplasmic reticulum. Recently, some types of congenital disorders of glycosylation (CDG) have been described as consequences of defects in dolichol biosynthesis and metabolism, yet these types of CDG are not detectable by standard screening methods. The aim of this project was to evaluate the potential of dolichol as a biomarker of CDG. Biological material for this study consisted of urine samples from 75 controls, 6 patients with CDG and 43 patients with suspicion of CDG; samples of the frontal cortex, liver, muscle and heart tissues from 2 patients with mutation in the NUS1 gene and controls. Molecular species profiles of dolichol were analyzed by liquid chromatography combined with tandem mass spectrometry. In the control group, a significant correlation between the ratio of dolichol 18 to dolichol 19 (Dol18/Dol19) and age was found in urine. We established a reference range for Dol18/Dol19 from urine samples. The ratio of Dol18/Dol19 was significantly higher in both urine and tissue samples from patients with mutation in NUS1 in comparison to controls. Our results show a novel diagnostic option for patients with rare congenital disorders of glycosylation. [ABSTRACT FROM AUTHOR]

• Over 500,000 infants screened in the past 5 years for Inborn errors of metabolism. • Tandem mass spectrometry and next-generation sequencing. • The incidence of Inborn errors of metabolism was 1:1941 in Jining. • PAHD and MMA were the two most common disorders. Inborn errors of metabolism (IEMs) can cause intellectual disability or even death in children. To evaluate the disease spectrum and genetic characteristics of IEMs in Jining City of Shandong Province in East China, we used tandem mass spectrometry (MS/MS) technology for IEMs screening combined with genetic analysis. Newborns were screened from July 14, 2014, to December 31, 2018. Amino acid and carnitine contents were detected by MS/MS. According to the results for normal newborns, the reference range of our laboratory was established with the percentile method. The suspected positive newborns were further diagnosed using next-generation sequencing. A total of 514,234 newborns were screened, and 265 were diagnosed with IEMs, with a detection rate of 1:1941. Of the 265 patients, 130 (49.06%) had organic acid disorders, 83 (31.32%) had amino acid disorders, 34 (12.83%) had fatty acid oxidation disorders, and 18 (6.79%) had urea circulatory disorders. PAHD and MMA were the two most common disorders. IEMs-associated genes were identified in 233 patients. Our data indicated that IEMs are never uncommon in Jining, and the disease spectrum and genetic background were clearly elucidated, contributing to the treatment and prenatal genetic counseling of these disorders in the region. [ABSTRACT FROM AUTHOR]

Gestational diabetes mellitus (GDM) is a common clinical disease with complicated clinical process and harmful effects on pregnant women and fetus. The purpose of this study is to use the convenient urine samples in combination with glucose levels to detect or predict GDM. In this study, urine samples of non-pregnant women, normal pregnant women and GDM patients were collected. The peptides in urine were enriched by weak cationic exchange magnetic beads (MB-WCX) and analyzed by matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS). 46 polypeptide peaks with statistical difference (P < 0.01) were screened out by using Bioexplorer analysis software. The level of molecules with mass-to-charge ratio of 1079.2, 1290.6 and 1500.7 was higher in the GDM group than the other two groups. Through the analysis of differential molecules by liquid chromatography tandem mass spectrometry (LC-MS), the above molecules were identified as coagulation factor IX, TBC1 family member 5 isoform a [Homo sapiens] (TBC1D5a) and immunoglobulin kappa constant. The discovery of polypeptides provides the research basis for further primary screening and assistant diagnosis of GDM through urine samples. [ABSTRACT FROM AUTHOR]

• A high-risk screening study was performed for patients with chronic kidney disease. • Globotriaosylceramide (Gb 3) was analyzed in dried urine spots by LC-MS/MS. • A total of 397 participants were recruited in four centers across Canada. • No Fabry patients were detected in this study. • Chronic kidney disease might be associated with increased urinary Gb 3 concentrations. Fabry disease is an X-linked lysosomal storage disorder with a highly heterogeneous clinical presentation. This complex disease is caused by a deficient activity of the enzyme α-galactosidase A, which is involved in the catabolism of glycosphingolipids. The prevalence of Fabry disease is underestimated, due to the presence of atypical variants. High-risk screening protocols are particularly relevant for this disease due to the availability of treatments, such as enzyme replacement and chaperone therapies. As kidney manifestations are present in the majority of male and many female patients with Fabry disease, a high-risk screening protocol was performed for patients with chronic kidney disease of unknown etiology. Recruitment of 397 participants took place in four centers across Canada from 2011 to 2017. Globotriaosylceramide (Gb 3) was analyzed in dried urine spots by liquid chromatography/tandem mass spectrometry followed by globotriaosylsphingosine (lyso-Gb 3) on the repeat analysis. The collection and shipment of urine specimens on filter paper resulted in easier handling/shipment and significant cost-saving. No Fabry patients were detected in this study. Increased concentrations of urinary Gb 3 were observed in 13.6% of patients with chronic kidney disease suggesting that chronic kidney disease or other comorbidities might be associated with increased urinary Gb 3 concentrations. [ABSTRACT FROM AUTHOR]

• First report on uremic toxins level in plasma and CSF of Parkinson's diseases (PD) • Correlation of uremic toxins with eGFR, hepcidin and oxidative stress was tested. • Patients with motor fluctuations had higher toxins level. • In PD higher ratio CSF/plasma for indoxyl sulfate and p-cresol sulfate was seen. • Uremic toxins can be associated with pathogenesis and progression of PD. In Parkinson's disease (PD), impairment of brain to blood barrier and/or blood-cerebrospinal fluid (CSF) barrier is described. It can increase the level of uremic toxins in CSF. So far, role of these compounds in neurological disorders has not been completely understood. However, a link has been observed between chronic kidney disease and neurological disorders. We measured the concentrations of uremic toxins (i.e. indoxyl sulfate (IS), p -cresol sulfate (pCS), symmetric dimethylarginine (SDMA), asymmetric dimethylarginine (ADMA), and trimethylamine N-oxide (TMAO)) in CSF and plasma, and correlated them with inflammation and oxidative stress biomarkers. Plasma and CSF samples were collected from 27 volunteers (18 with PD and 9 controls). The level of toxins was determined using liquid chromatography coupled with tandem mass spectrometry. In PD, for IS and pCS, CSF-plasma ratio was higher. Concentration of pCS in CSF was higher in PD compared to controls. TMAO level was also higher in plasma of that group. Patients with motor fluctuations had higher level of uremic toxins in CSF, but not in plasma. The level of pCS and IS in CSF of PD is higher than expected, based on their blood level. It can influence pathogenesis and progression of PD. [ABSTRACT FROM AUTHOR]

• The total concentration of Lyso-Gb 3 plus its analogues in urine, is specific to FD and may provide extra diagnostic utility for both classical and non-classical FD patients. • The total concentration of Lyso-Gb 3 plus its analogues in urine, enhances the diagnostic sensitivity to 100% and helps to avoid any overlap between the levels in FD patients and healthy control subjects. Fabry disease (FD) is a genetic disorder caused by defective α-galactosidase-A enzyme due to mutations in the GLA gene. A reliable diagnosis in classical FD males can be made by measuring the enzyme activity while diagnosing classical FD females and non-classical FD patients requires mutation analysis. Plasma globotriaosylsphingosine (Lyso-Gb 3) has progressively gained more importance as a diagnostic biomarker for FD in recent years. Having another biomarker to complement plasma Lyso-Gb 3 will increase the diagnostic accuracy in the era of mass screening, and also precision medicine. This study aims to highlight the clinical utility of the total concentration of urinary Lyso-Gb 3 plus its analogues in diagnosing FD. Random urine samples collected from 42 FD patients and 48 healthy individuals. Lyso-Gb 3 and its analogues were enriched by solid phase extraction and analysed by liquid chromatography tandem mass spectrometry. The total concentration of Lyso-Gb 3 plus its analogues in classical FD male and female patients were 1124.4 ± 181.2 and 308.6 ± 78.6 pmol/mmol creat., respectively. The levels in non-classical FD male and female patients were 229.2 ± 169.4 and 314.4 ± 156.4 pmol/mmol creat., respectively. Urinary Lyso-Gb 3 and its analogues were virtually undetectable in healthy volunteers making it 100% specific for FD diagnosis. For FD male and female patients, total concentration of urinary Lyso-Gb 3 plus its analogue levels ≥0.25 pmol/mmol creat. yielded a diagnostic sensitivity and specificity of 100% (AUC = 1, p < 0.00001). Our study findings support that the total concentration of Lyso-Gb 3 plus its analogues in urine is specific to FD and may provide extra diagnostic utility for both classical and non-classical FD patients. [ABSTRACT FROM AUTHOR]

Therapeutic drug monitoring of azathioprine metabolites is required for pharmacotherapy individualisation in patients with inflammatory bowel disease. Currently mainly hemolysates are used, requiring long sample preparation and showing limited analytes stability. Therefore, a quantitative LC-MS/MS method for determination of 6-thioguanine (6-TG) and 6-methylmercaptopurine (6-MMP) in dried blood spot samples (DBS) was developed. Analysis involves liquid extraction from 30 μL blood spot, hydrolysis and quantification with LC-MS/MS. Method met the validation criteria in terms of selectivity, linearity, accuracy, and precision in a range from 50 to 5300 pmol/8 × 108 Ery for 6-TG and from 260 to 5300 pmol/8 × 108 Ery for 6-MMP. Range can be increased to 8000 pmol/8 × 108 Ery. No matrix effect was observed and the recovery was >80%. DBS specific validation parameters were confirmed: spot homogeneity, no influence of blood spot volume (>30 μL) on 6 mm DBS disk, and absence of haematocrit effect. DBS samples were stable for at least one month at temperatures from −20 to 40 °C. Clinical validation confirmed that DBS method and routine clinical method with hemolysate samples give comparable results and enable similar clinical decisions. The newly developed DBS method is simple and presents an alternative to conventional methods for therapeutic drug monitoring of azathioprine metabolites. • DBS method for measuring 6-TG and 6-MMP levels in patients with IBD was developed. • Analysis involves liquid extraction, hydrolysis and quantification with LC-MS/MS. • Method meets general and DBS specific validation criteria. • DBS method and routine method using hemolysate enable similar clinical decisions. • New DBS method is simple and presents an alternative to conventional methods. [ABSTRACT FROM AUTHOR]

In this study, we evaluated the incidence and genetic characteristics of hyperphenylalaninemia (HPA) in Xiamen, China. We analyzed the newborn screening data of HPA, obtained using a fluorometric method and tandem mass spectrometry (MS/MS), from 2013 to 2017. The suspected positive samples were further diagnosed using MassArray technology, multiplex ligation-dependent probe amplification (MLPA), and Sanger sequencing. A total of 418,831 newborns were screened, of whom 19 were diagnosed as HPA patients, with an incidence of 1:22,044. Of these HPA patients, 15 tested positive for phenylketonuria (PKU, 1:27922), and 4 tested positive for tetrahydrobiopterin deficiency (BH4D, 1:104,708). A total of 17 mutations were identified among 38 alleles in the 19 patients, with a detection rate of 94.74%, including 13 PAH and 4 PTS mutations. Among these, the c.721C>T, c.728G>A, c.1197A>T, c.611A>G and c.331C>T mutations, and the c.259C>T and c.155A>G mutations were the most prevalent PAH and PTS mutations in Xiamen, respectively. Therefore, this study systematically demonstrated the incidence and mutation spectrum of HPA in Xiamen. This information would contribute to genetic counseling, prenatal diagnosis, and management of HPA patients. Moreover, combining MS/MS technology with molecular genetic diagnosis is an effective strategy for future newborn HPA screening in Xiamen. • The incidence of hyperphenylalaninemia (HPA) in Xiamen is about 1 in 22,044 newborns. • The overall incidence of phenylketonuria (PKU) is 1:27922 in Xiamen. • Tetrahydrobiopterin deficiency (BH4D) has an incidence of 1:104,708 in Xiamen. • The study has clearly elucidated the mutations of PAH and PTS gene in this region. [ABSTRACT FROM AUTHOR]

Immunoassays and liquid chromatography tandem mass spectrometry (LC-MS/MS) are two major methods for therapeutic drug monitoring (TDM) of immunosuppressant drugs. Compared to the relatively limited analytical performance and cross reactivities of immunoassays, the LC-MS/MS method is considered as a gold standard; however, the lack of systematic evaluation and standardization needs to be addressed. A LC-MS/MS method for the determination of cyclosporine A, sirolimus, tacrolimus, and everolimus was developed. One-step protein precipitation was used to prepare blood samples. The newly developed method was systematically evaluated and validated according to the standard guidelines. The quantitative method for four immunosuppressant drugs in human whole blood was validated according to the guidelines. The lower limits of the measuring interval (LLMI) for cyclosporine A, sirolimus, tacrolimus, and everolimus were 5, 0.5, 0.5, and 0.5 ng/mL, respectively. Linear correlation coefficients were all >0.999. Internal standard-normalized (IS-normalized) matrix correction factor was within the range 0.88–1.17. The average spiked recoveries of five replicates for the four immunosuppressant drugs were in the range 87.4–109.6%. An LC-MS/MS method combined with one-step protein precipitation was developed, providing short sample preparation and chromatographic run time, thus allowing easy clinical diagnosis. • A straightforward LC-MS/MS method was developed for determination of four immunosuppressant drugs. • One-step protein precipitation was used to deal with the blood samples, which was easy and high throughput. • Systematic evaluation were validated to access the method strictly according to the guidelines [ABSTRACT FROM AUTHOR]

Melatonin is one of numerous biologically active compounds reported to be stored in platelets. As melatonin is increasingly linked to several diseases, we wanted to confirm the storage of melatonin in platelets using sensitive liquid chromatography in combination with isotope dilution tandem mass spectrometry (LC-MS/MS). The difference between melatonin levels analyzed in platelet-rich plasma (PRP) and platelet-poor plasma (PPP) served as proxy for platelet levels of melatonin. Melatonin concentrations were analyzed in PRP and PPP from nineteen healthy volunteers by ELISA and LC-MS/MS. A Wilcoxon signed-rank test was performed to assess if the melatonin levels measured in PRP and PPP were different. Results for melatonin concentrations obtained by LC-MS/MS or ELISA were compared using Passing-Bablok regression. Comparison of the ELISA with the LC-MS/MS method showed poor agreement for melatonin concentrations in PRP and PPP. No indication was found for storage of melatonin in platelets by either LC-MS/MS or ELISA (P =.89 and P =.53 for the LC-MS/MS and ELISA analysis, respectively). In this study we could find no evidence for melatonin storage in platelets. • No evidence found for storage of melatonin in platelets • Poor agreement between ELISA and LC-MS/MS assay for plasma melatonin concentrations [ABSTRACT FROM AUTHOR]

• The rare variant c.1663G>A is most frequent in Henan population. • The infants carrying the c.1663G>A variant tend to be responsive to vitamin B12 and have a good prognosis. • Five novel variants (c.479C>T, c.811G>C, c.965T>A, c.1142G>A and c.1667C>T) are identified in this study. Methylmalonic acidemia (MMA) is the most common organic acidemia in China, and isolated MMA accounts for approximately 30 % of all types of MMA. Common variants of the MMUT gene vary greatly around the world. The present study aims to determine the high-frequency and novel genetic variants of the MMUT gene in the Henan population of China and evaluate the prognosis of patients carrying the c.1663G>A (p.Ala555Thr) variant. We performed next-generation sequencing for 41 patients with isolated MMA screened by tandem mass spectrometry (MS/MS) and analysed the genetic results. We also evaluated the prognosis of patients with the c.1663G>A variant. We used Jalview software for multispecies sequence alignment and Missense3D and DynaMut to predict the protein function of the detected novel variants. A total of 43 variants from 41 patients with isolated MMA were detected, of which c.1663G>A (14.63 %), c.729_730insTT (10.98 %), and c.1106G>A (8.53 %) are high-frequency variants of the MMUT gene in the Henan population. The patients carrying the c.1663G>A variant tended to be responsive to vitamin B12, have a low mortality rate. We also identified 5 novel variants (c.479C>T, c.811G>C, c.965T>A, c.1142G>A and c.1667C>T). The rare variant c.1663G>A is prevalent in the Henan population, and infants with this variant tend to have good prognosis. Our findings, especially novel variants, will help broaden the spectrum of genetic variants and facilitate clinical diagnosis and genetic counselling for affected families. [ABSTRACT FROM AUTHOR]

• The difference in the efficacy of lenalidomide is related to metabolites. • Ultra-performance liquid chromatography–time-of-flight tandem mass spectrometry (UPLC-TOF-MS/MS) is used to detect the metabolic. • Eleven metabolites are identified and characterized, including 4 novel compounds. • Two metabolites: Denitrified-2 glutarimide and 5-hydroxylenalidomide participate in the efficacy. Lenalidomide is the first-line drug for the clinical treatment of multiple myeloma. However, its efficacy differs significantly among patients. Clinically, after lenalidomide treatment, few patients' conditions worsened, whereas others remained stable or improved. To clarify the reasons for this difference in efficacy, 20 patients with multiple myeloma who received maintenance treatment with lenalidomide were retrospectively included in this study. Lenalidomide metabolic compounds were detected in patient urine using mass spectrometry. A rapid and accurate ultra-performance liquid chromatography–time-of-flight tandem mass spectrometry (UPLC-TOF-MS/MS) method was used to characterize metabolites in the urine of different patients. Eleven metabolites, including four new compounds, were identified and characterized in all the samples. Among these, two metabolites were found to have obvious discrepancies in different groups of patients. One metabolite named Denitrified-2 glutarimide, a new potential compound, was only detected in the urine of ineffective and stable patients, whereas the other metabolite named 5-Hydroxy-lenalidomide was found only in the urine of effective patients. [ABSTRACT FROM AUTHOR]

• Dried blood spot (DBS) sampling is a sustainable alternative to venipuncture which can be performed at home. • A highly accurate and rapid assay for the simultaneous quantification of vancomycin and creatinine in DBS using LC-MS/MS was successfully developed and validated. • Hematocrit levels between 0.3 and 0.5 L/L do not affect the accuracy of vancomycin and creatinine measurements in DBS. • Vancomycin and creatinine are stable in DBS at room temperature and in the freezer −20 °C for at least four weeks. Vancomycin is a widely used antibiotic for the treatment of gram-positive bacterial infections, especially for methicillin-resistant Staphylococcus aureus (MRSA) infections. Due to a small therapeutic range and large inter-patient variability, therapeutic drug monitoring (TDM) of vancomycin is required to minimize toxicity and maximize treatment efficacy. Venous blood sampling is mostly applied for TDM of vancomycin, although this widely used sampling method is more invasive compared to less painful alternatives, such as the dried blood spot (DBS) method, which can be performed at home. We developed an UPLC-MS/MS method for the quantification of vancomycin and creatinine in DBS. A fast sample preparation and short analysis run time of 5.2 min were applied, which makes this method highly suitable for clinical settings. Validation was performed according to international (FDA and EMA) guidelines. The validated concentration range was found linear for creatinine from 41.8 µmol/L to 722 µmol/L and for vancomycin from 3.8 mg/L to 76.6 mg/L (r2 > 0.990) and the inaccuracies, imprecisions, hematocrit effects, and recoveries were < 15 % for both compounds. No significant carryover effect was observed. Hence, we successfully validated a quantification method for the simultaneous determination of creatinine and vancomycin in DBS. [ABSTRACT FROM AUTHOR]

• UHPLC-MS/MS procedures were developed to measure total and unbound plasma antiretroviral concentrations. • Antiretrovirals evaluated were bictegravir, darunavir, doravirine, and dolutegravir. • Unbound concentrations were obtained by combined equilibrium dialysis and UHPLC-MS/MS procedures. • The procedures' applicability was verified using subjects receiving antiretroviral therapy. • The measurement procedures can be helpful for therapeutic drug monitoring of antiretrovirals in routine laboratories. Fixed-dose combinations of antiretroviral drugs are commonly used to treat HIV infection and therapeutic monitoring is not part of routine clinical practice. However, drug concentrations monitoring might have role in different clinical scenarios as well as for research purposes. This study aimed to develop and validate UHPLC-MS/MS procedures for measuring total and unbound concentrations of bictegravir, dolutegravir, darunavir and doravirine in human plasma. Equilibrium dialysis preceded sample preparation (based on protein precipitation) for measuring unbound antiretroviral concentrations. Chromatographic separations were achieved on an Acquity®-UPLC® HSS™-T3 column (50 mm × 2.1 mm; 1.8 µm) using a non-linear water/acetonitrile gradient containing 0.1 % formic acid at a 0.5 mL/min flow rate. Antiretrovirals were detected by tandem mass spectrometry in positive electrospray ionisation and multiple reaction monitoring modes. No significant interferences or carry-over were observed. Imprecisions, absolute relative biases, normalised matrix effects and recoveries were ≤15.0 %, ≤11.1 %, (94.7–104.1)% and (96.7–105.5)%, respectively. Non-linear measuring intervals were observed between (25–10,000) µg/L for total/plasma dialysate concentrations and linearity schemes (1.00–100) µg/L for buffer dialysate concentrations. The UHPLC-MS/MS procedures developed could be used for research purposes and therapeutic drug monitoring of antiretrovirals in routine clinical practice. [ABSTRACT FROM AUTHOR]

• Nine infants with the same genotype presented different CIT concentrations. The relationship between the CIT level and genotype is uncertain. • Four variations, c.851_854delGTAT, c.615 + 5G > A, c.1638_1660dup and IVS16ins3kb, constitute the high-frequency mutations in Quanzhou. • Patient No. 22 possessed compound heterozygous mutations in the SLC25A13 gene accompanied by compound heterozygous variations in the PTS gene. This is the first reported case of the simultaneous occurrence of tetrahydrobiopterin (BH4) deficiency and NICCD. • Patient No. 26 is the first reported case of the simultaneous occurrence of primary carnitine deficiency (PCD) and NICCD. • Combining genetic analysis with the routine NBS program is emphasized. Neonatal intrahepatic cholestasis due to citrin deficiency (NICCD) is an autosomal recessive disorder caused by SLC25A13 genetic mutations. We retrospectively analyzed 26 Chinese infants with NICCD (years 2014-2022) in Quanzhou City. The plasma citrulline (CIT) concentration analyzed by tandem mass spectrometry (MS/MS), biochemical parameters and molecular analysis results are presented. Twelve genotypes were discovered. The relationship between the CIT concentration and genotype is uncertain. In total, 8 mutations were detected, with 4 variations, c.851_854delGTAT, c.615 + 5G > A, c.1638_1660dup and IVS16ins3kb, constituting the high-frequency mutations. Specifically, we demonstrated 2 patients with NICCD combined with another inborn errors of metabolism (IEM). Patient No. 22 possessed compound heterozygous mutations of c.615 + 5G > A and c.790G > A in the SLC25A13 gene accompanied by compound heterozygous variations of c.C259T and c.A155G in the PTS gene. Additionally, Patient No. 26 carried c.51C > G and c.760C > T in the SLC22A5 gene as well as c.615 + 5G > A and IVS16ins3kb in the SLC25A13 gene. We report a case of the simultaneous occurrence of primary carnitine deficiency (PCD) and NICCD. [ABSTRACT FROM AUTHOR]

The current review provides an overview on the development of 25(OH)D measurement standardization tools over the last three decades and clarifies whether there is a role as a serum biomarker for vitamin D in neurological diseases. In the past, a lack of internationally recognized 25(OH)D reference measurement procedures and reference standard materials led to unstandardized serum total 25(OH)D results among research and clinical care laboratories. The vitamin D Standardization Program (VDSP) has been introduced in 2010 to address this problem, however, vitamin D External Quality Assessment Scheme (DEQAS) reports still show substantial sample- to- sample variability. Further, immunoassays, which are mainly used in clinical care laboratories, display analytical issues, including matrix-effects interferences, which cannot be overcome by the standardization process. Hence, liquid chromatography-tandem mass spectrometry (LC/MS-MS) methods should be used to measure 25(OH)D. Low vitamin D serum levels have been found in patients affected by Alzheimer's disease and Parkinson's disease, suggesting a role for vitamin D as a serum biomarker in these diseases. However, few studies reported 25(OH)D standardized results, thus, no clear evidence on the potential role of 25(OH)D serum levels in these diseases exists. • There is no evidence-based consensus on which 25(OH)D values define Vitamin D insufficiency, deficiency and sufficiency. • Recent DEQAS reports show substantial sample- to- sample variability, especially using immunoassays. • LC/MS-MS method shows the best performance for measuring 25(OH)D in terms of accuracy, compared to immunoassays. • Few studies on Alzheimer Disease and Parkinson Disease patients reported 25(OH)D standardized data. • No clear evidence on the role of 25(OH)D serum levels as a useful biomarker in neurological diseases is available. [ABSTRACT FROM AUTHOR]

Evidence is accumulating which may result in plasma free metadrenalines (PMets) becoming the preferred test for diagnosing phaeochromocytoma and paraganglioma. Moreover, increased availability and benefits over other analytical methods like liquid chromatography with electrochemical detection and immunoassay are causing liquid chromatography tandem mass spectrometry (LC-MS/MS) to become the method of choice for PMet measurement. This review explores the evidence-base supporting this, and summarises published LC-MS/MS analytical methods for PMet analysis. Key aspects of methods (including SPE extraction, HILIC chromatography, MRM MS-detection and standardisation) are discussed. Common causes of analytical interference (e.g. ion suppression/enhancement, ionic cross talk, in source transformation and isobaric interferences) are outlined to illustrate the importance of sample purification and chromatographic resolution. The importance of supine, fasting sampling and Bayesian interpretation against supine, fasting reference intervals are explained, as well as the importance of age-specific reference intervals for normetadrenaline. Confounding factors like diet, drugs, renal function and acute illness are explored, along with potential strategies to address these (e.g. CKD-specific reference intervals). • Advantages of plasma free metadrenalines (PMets) over urine metadrenalines. • Advantages of liquid chromatography tandem mass spectrometry (LC-MS/MS) measurement of PMets over liquid chromatography with electrochemical detection and immunoassay. • Summary of published LC-MS/MS analytical methods for PMet analysis. • Summary of factors to be considered when interpreting PMet results (including analytical interference, reference intervals, diet, drugs, renal function and acute illness). [ABSTRACT FROM AUTHOR]

Carnitine-acylcarnitine translocase deficiency (CACTD) is a rare autosomal recessive disorder of mitochondrial fatty acid oxidation that occurs due to mutations in the SLC25A20 gene. Severe CACTD results in neonatal or infantile sudden death. Herein, we reported six patients with CACTD diagnosed based on biochemical and molecular findings from 5 unrelated families in Guangdong from 2016 to 2017. Among them, five patients presented with hypotonia, nonketotic hypoglycemia, and arrhythmia 2 days after birth, while the other patient presented with respiratory distress, hypotonia, and arrhythmia. Five of the patients died in the neonatal period. Blood acylcarnitine concentrations determination from dried blood spots (DBS) were measured by tandem mass spectrometry (MS/MS). The SLC25A20 and CPT2 gene sequences were analyzed by direct Sanger sequencing. SLC25A20 gene analysis revealed a c.199-10T>G (IVS2-10T>G) homozygous variants in four unrelated patients and a novel mutation c.199-10T>G/c.719-8_c.719-1dupCCCACAG compound heterozygous variants in twins. This report describes the clinical characteristics, biochemical findings and molecular analysis of SLC25A20 gene of patients with CACTD in Guangdong. And our results show that the c.199-10T>G is likely the most common variant of CACTD in Guangdong population as it accounts for 83% (10/12) of the observed mutant alleles. Individuals with the c.199-10T>G genotype had a severe CACTD phenotype. • We reported six patients with CACTD diagnosed based on biochemical and molecular findings from 5 unrelated families in Guangdong and shared their meaningful family history. • We identified a novel c.719-8_c.719-1dupCCCCACAG variant that probably affects the splicing predicted by several in silico prediction tools. • The diagnostic process of CACTD was summarized based on clinical analysis and literature review in order to provide effective reproductive counseling to couples. [ABSTRACT FROM AUTHOR]