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In recent years, scientific researchers have increasingly become interested in noninvasive sampling methods for therapeutic drug monitoring and disease diagnosis. As a result, dried saliva spot (DSS), which is a sampling technique for collecting dried saliva samples, has been widely used as an alternative matrix to serum for the detection of target molecules. Coupling the DSS method with a highly sensitive detection instrument improves the efficiency of the preparation and analysis of biological samples. Furthermore, dried blood spots, dried plasma spots, and dried matrix spots, which are similar to those of the DSS method, are discussed. Compared with alternative biological fluids used in dried spot methods, including serum, tears, urine, and plasma, saliva has the advantage of convenience in terms of sample collection from children or persons with disabilities. This review aims to provide integral strategies and guidelines for dried spot methods to analyze biological samples by illustrating several dried spot methods. Herein, we summarize recent advancements in DSS methods from June 2014 to March 2021 and discuss the advantages and disadvantages of the key aspects of this method, including sample preparation and method validation. Finally, we outline the challenges and prospects of such methods in practical applications.

Aspirin is a widely used anti-inflammatory drug. It is reported that a relationship may exist between salicylic acid content in plasma and saliva after taking aspirin. This study established a rapid, convenient, and safe method to assess salicylic acid concentration in human saliva. A novel HPLC-ultraviolet detector was used to measure salicylic acid concentrations in human saliva and plasma. A C18 reversed-phase column with an aqueous solution of 0.1% trifluoroacetic acid (TFA)-acetonitrile mobile phase was used, and drug peaks were recorded at 303 nm. Salicylic acid was completely separated in saliva and plasma. Excellent linearity and correlation (r

We developed a novel fluorescent chiral probe, DBD-trans-2-methyl-L-proline (DBD-M-Pro), which can be used to target recognition of amino functional groups using chiral resolution. To investigate the chiral resolution efficiency, 20 chiral amino enantiomers (19 DL-amino acids and phenylethylamine) were labeled using ultra-performance liquid chromatography (UPLC) with a fluorescence (FL) system. Diastereomers were formed by the reactions of DBD-M-Pro with enantiomers of amino functional groups at 60°C for 60 min and detected on a BEH C18 column (100 × 2.1 mm, 1.7 μm). Gradient elution of 10 mM ammonium acetate with 0.05% formic acid (FA) aqueous solution and 0.1% FA acetonitrile or 0.1% FA methanol solution was performed at an excitation wavelength (Ex) 460 nm and emission wavelength (Em) 550 nm. Each resulting derivative of D- and L- type was effectively separated. The results showed that the resolution (Rs) of 17 amino acids and phenylethylamine (PEA) in the range of 1.59–24.11, except for histidine (His) (Rs=1.32) and serine (Ser) (Rs=1.47), achieved completely separation. The DBD-M-Pro chiral probe has a robust chiral selectivity for D-amino acids. Furthermore, a new method for the simultaneous determination of six DL-amino acids (Pro, Val, Trp, Phe, Leu, Lys) in human saliva was developed. The proposed method showed resolution values of 1.78–16.38, and an excellent linear relationship was obtained in the range of 2.5–500 pmol (R2≥0.9990). The limit of detection (S/N=3) ranged from 0.5 to 3.75 pmol. The intra-day and inter-day coefficient of variation (CV) were within the range of 1.75%–11.73%. The average addition recoveries in saliva ranged from 95.99% to 106.97%. The methodology was used to determine the content of DL-amino acids and the D/L-amino acid ratio in the saliva of 40 healthy volunteers (15 males and 25 females), as well as evaluating the differences between men and women. Our study suggests that the DBD-M-Pro chiral probe could be an effective tool for screening potential D-amino acid biomarkers in disease.

A novel derivatization reagent, N -[1-Oxo-5-(triphenylphosphonium)pentyl]-( S )-3-aminopyrrolidine (OTPA), with triphenylphosphine (TPP) as a basic structure carrying a permanent positive charge was developed for the enantiomeric separation of chiral carboxylic acids by high-performance liquid chromatography (HPLC). OTPA reacted with the carboxylic acids at 40 °C within 90 min in the presence of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and 1-hydroxybenzotriazole (HOBt). The degree of epimerization (racemization) during the derivatization reaction was negligible. The separability of the diastereomers was evaluated in terms of their resolution value (Rs). The Rs values of the derivatives of non-steroidal anti-inflammatory drugs (NSAIDs), which were selected as the representative carboxylic acids, were completely separated by reversed-phase chromatography using an ODS (4.6 mm × 150 mm I.D., 5.0 μm) column. The resolution Rs values were 1.54–2.23 for the evaluated carboxylic acids and the OTPA-derivatization was also effective for the enantiomeric separation of chiral carboxylic acids. The calibration curves (r 2 > 0.9971) were linear over the concentration range of 0.0125–1.25 mM for each enantiomer of ketoprofen (KET), and naproxen (NAP), 0.05–1.0 mM for each enantiomer of ibuprofen (IBU), 2-phenylpropionic acid (PPA), and loxoprofen (LOX), and 0.05–1.25 mM for each enantiomer of PBA. The limit of detection (S/N = 3) for each of the enantiomers of the NSAIDs and chiral carboxylic acid enantiomers was 1.4–7.6 μmol/L. The inter-day and intra-day assay precisions were all

Measurement of chiral thiol compounds such as glutathione (GSH), cysteine (Cys), and homocysteine (Hcy) in human serum plays an important role in the early diagnosis and warning of cardiovascular disease, neurodegenerative disease, and cancer. We developed a novel chiral mass spectrometry derivatization reagent, (R)-(5-(3-isothiocyanatopyrrolidin-1-yl)-5-oxopentyl) triphenylphosphonium (NCS-OTPP), with triphenylphosphine (TPP) as a basic structure carrying a permanent positive charge for the diastereomeric separation of chiral thiol compounds by ultrahigh-performance liquid chromatography coupled to quadrupole-Orbitrap high resolution mass spectrometry (UHPLC-Q-Orbitrap HRMS). A novel method was developed for simultaneous determination of three kinds of chiral thiol compounds based on the NCS-OTPP derivatization method. Three kinds of chiral thiol compounds on a YMC Triart C18 (2.0 × 150 mm, 1.9 μm) column with Rs were 1.56–1.68. The protonated precursor to product ion transitions monitored for GSH was m/z 780.16→747.24/473.18, Cys was m/z 594.20→561.18/473.18, and Hcy was m/z 608.21→575.19/473.18. An excellent linearity for all the analytes with correlation coefficients ≥ 0.9995 and suitable precision with inter-day and intra-day coefficients of variation RSDs was 0.83–4.06% and 0.95–3.11%. Satisfactory accuracy with recoveries between 83.73 and 103.35% was observed. The limit of detection (S/N = 3) was 2.4–7.2 fmol. Furthermore, the method was successfully applied to the simultaneous determination of three kinds of free and total thiol compounds in serum from 10 healthy volunteers at normal and stress states.

A high-sensitivity and -selectivity mass spectrometry derivatization reagent, (R)-(5-(3-isothiocyanatopyrrolidin-1-yl)-5-oxopentyl) triphenylphosphonium (NCS-OTPP), was developed for the enantiomeric separation of chiral thiol compounds as prospectively important diagnostic markers for oxidative stress-related diseases. Complete separation of GSH, DL-Cys, and DL-Hcy was achieved. The parent ions of all derivatives had a fragment of m/z 473.18 and a structure of m/z 75.95 (R-S = C-S-R'), conducive to qualitative and quantitative analysis. Good linear relationships were obtained for all analytes (R2≥ 0.9995). The intra-day and inter-day precision were 0.82-5.16 % and 1.02-4.18 % in saliva, and 0.81-3.45 % and 0.99-6.47 % in urine, with mean recoveries of 83.31-105.66 % and 84.09-101.11 %, respectively. The limit of detection (S/N = 3) was 19.20-57.60 nM. Free and total GSH, DL-Cys, and DL-Hcy were detected simultaneously in saliva and urine from 10 volunteers in the normal, stressed, and stable states by UHPLC-Q-Orbitrap HRMS. The thiol compounds were quantitatively related to oxidative stress state changes.

Concentration of uric acid (UA) in serum is one of the markers used to diagnose gout and hyperuricemia. However, serum treatment and storage are cumbersome, and wounds are susceptible to infection. Therefore, a new sampling and analysis method using noninvasive biological samples has been developed, called the dried spot method of UA in human saliva (DSM-UHS). Saliva (5 μL) was dropped on filter paper (a spot with a diameter of 5 mm) containing hypoxanthine (IS) (5 μL) and dried at room temperature for 30 min. The filter paper was immersed in 200 μL of lithium carbonate solution and shaken in a block bath shaker for 5 min at 30 °C. Afterward, the extraction was concentrated and reconstituted with 100 μL of lithium carbonate solution analyzed by HPLC-UV. When comparing the concentration of UA in the human saliva of hyperuricemia patients (HPs) and with that of healthy volunteers (HVs), we observed the concentration of UA was higher in the HPs than in the HVs (p 0.0001). In addition, the results showed a significant linear relationship between the content of UA in saliva and the content of UA in the serum (r = 0.6243). The content of UA in human saliva could indirectly reflect the content of UA in human serum. Then DSM-UHS could be used to determine the content of UA in the saliva of HVs and HPs. This study provides a new research method and strategy for the determination of human UA content and the clinical prewiring of hyperuricemia.

A method for the determination of D-Aspartic acid (D-Asp) and its D/L ratio in peptides and proteins has been developed. This method was carried out with good separation of the D/L chiral peptide pairs by combination of a chiral derivatization and an ADME column separation. Furthermore, a cationic derivatization reagent, DBD-Py-NCS, increased the sensitivity of the ESI-MS/MS detection. To confirm the comprehensive peptide analysis, synthesized α-Crystallin tryptic peptides, which included D-Asp residues, were analyzed. The 5 pairs of D/L-Asp that included peptide diastereomers were well separated. Their peak resolutions were more than 1.5 and the results were reproducible (RSD0.05, n=5). As an application of this method, we analyzed the α-Crystallin standard and UV irradiated α-Crystallin. After trypsin digestion and DBD-Py-NCS derivatization, the tryptic peptide derivatives were applied to LC-MS/MS. Based on the results of peptide sequence identification, almost all the tryptic peptides of the αA- and αB-Crystallin homologous subunits of α-Crystallin were detected as DBD-Py NCS derivatives. However, there was no D-Asp residue in the standard proteins. In the case of the UV irradiated α-Crystallin, Asp

We have developed three kinds of novel derivatization reagents (4-CEBTPP, 4-CBBTPP, 5-COTPP) with triphenylphosphine (TPP) as a basic structure carrying a permanent positive charge for resolution of the oligosaccharides in glycoprotein using high-performance liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). The synthesized reagents reacted with the sialylglycosylamine of the sialylglycopeptide after treatment by PNGase F. The final derivatives were analyzed by ESI-MS and sensitively detected in the selected reaction monitoring (SRM) mode. Furthermore, the limits of detection (S/N=3) on the SRM chromatograms were at the fmol level (30fmol). Therefore, we used the limit of detection of the reagent products detected by the SRM and evaluated the utility of each reagent. Among the reagents, the positively charged 4-CEBTPP derivative's peak area was the highest; 4-CEBTPP with a positively charged structure showed about a 20 times greater sensitivity for the glycosylamine of the SGP product compared to the conventional fluorescence reagent, Fmoc-Cl. In addition, various fragment ions based on the carbohydrate units also appeared in the MS/MS spectra. Among the fragment ions, m/z 627.37 (CE=40eV) corresponding to 4-CEBTPP-GlcNAc and m/z 120.09 (CE=100eV) corresponding to 4-CEBTPP are the most important ones for identifying the oligosaccharide. 4-CEBTPP-SGA was easily identified by the selected-ion chromatogram in the product ion scan (m/z 120.09) and in the precursor ion scan (m/z 627.37) by MS/MS detection. The derivatized analytes have a high ionization efficiency and they are detected with a high sensitivity in the electrospray ionization. The novel derivatization reagent with a multi-function provided a higher sensitivity for the oligosaccharide analysis, as well as a better specificity and feasibility. Furthermore, several oligosaccharides in fetuin and ribonuclease B were successfully identified by the proposed procedure.

This study presents two simple and rapid methods for the quantification of therapeutic mAbs based on LC. Two mAbs (bevacizumab and infliximab) in plasma samples were purified using magnetic beads immobilized with a commercially-available idiotype antibody for each mAb. Purified mAbs were separated with HT-RPLC and detected with their native fluorescence. Using immunoaffinity beads, each mAb was selectively purified and detected as a single peak in the chromatogram. The HT-RPLC achieved good separation for the mAbs with sharp peaks within 20 min. The calibration curves of the two mAbs ranged from 1 to 20 μg mL(-1) (bevacizumab) and 1-10 μg mL(-1) (infliximab), and they had strong correlation coefficients (r(2) > 0.998). The LOD of bevacizumab and infliximab was 0.07 and 0.15 μg mL(-1), and the LLOQ of bevacizumab and infliximab was 0.12 and 0.25 μg mL(-1), respectively. Thus, the sensitivities were sufficient for clinical analysis. Immunoaffinity purification with HT-RPLC produced a selective and accurate bioanalysis without an LC-MS/MS instrument. Both methods could become general-purpose analytical methods and complement the results obtained with conventional LBA.

A rapid and simple analytical method for the quantification of uric acid (UA) in human fingernails by high-performance liquid chromatography (HPLC) with ultraviolet (UV) detection is described. UA was extracted from human fingernail samples at 90°C for 20min, then separated on an Inertsil ODS-2 column (250×4.6mm I.D., 5.0μm, GL Sciences) by isocratic elution using methanol: 74mM phosphate buffer (pH 2.2) 2:98 (v/v). An UV detector was used to monitor at 284nm. The results indicated that under optimized measurement conditions results were achieved within 8.0min, and a good linearity was achieved from the calibration curves (r(2)>0.9999) in the range of 1.0-10000ng; the limit of detection (S/N=3) was 2.0pg, the inter-day and intra-day assay precisions were all less than 0.46% and the mean recoveries (%) of the uric acid spiked in the human fingernail were 101.95%. The amounts of UA in the fingernails of healthy volunteers were determined.

In this study including the field of metabolomics approach for food, the evaluation of untargeted compounds using HILIC-ESI/TOF/MS and multivariate statistical analysis method is proposed for the assessment of classification, contamination and degradation of infant formula. HILIC mode is used to monitor more detected numbers in infant formulas in the ESI-positive scan mode than the reversed phase. The repeatability of the non-targeted contents from 4 kinds of infant formulas based on PCA was less than the relative standard deviation of 15% in all groups. The PCA pattern showed that significant differences in the classification of types and origins, the contamination of melamine and the degradations for one week were evaluated using HILIC-ESI/TOF/MS. In the S-plot from the degradation test, we could identify two markers by comparison to standards as nicotinic acid and nicotinamide. With this strategy, the differences from the untargeted compounds could be utilized for quality and safety assessment of infant formula.

Dicyandiamide is a compound for reducing the negative effects of greenhouse gas emissions and nitrate leaching into waterways. In this study, the trace contamination of dicyandiamide in infant formula was analysed by stable isotope dilution hydrophilic interaction liquid chromatography with tandem mass spectrometry (HILIC–MS/MS). Dicyandiamide and a stable isotope internal standard were monitored by multiple reaction-monitoring with mass transitions: m/z 85 → 68/43 and m/z 89 → 71/45 in the electrospray positive ion mode. For sample preparation of the infant formula, a diluted/filtered procedure was developed for this assay. The calculated LOD and LOQ values were 0.01 or 0.05 ng/mL for the standard solution, respectively. The averaged recovery and precision were 110.8% and 7.4%, respectively. This assay was applied to monitor 23 infant formulas, and the dicyandiamide contamination in one sample was detected and quantified at 79.1 ± 1.2 ng/g (ppb) powder. We suggest that it is necessary to cautiously monitor the DCD in common products from international countries.

Background In diabetes mellitus (DM) patients with ketoacidosis, ketone bodies, i.e., acetone, acetoacetic acid (AA) and β-hydroxybutyric acid (HA), are increased in the blood and urine. Acetone is also excreted by breathing due to the spontaneous decomposition of AA. Thus, the increase in acetone has been considered as one of the biomarkers for the diagnosis of DM. However, the determination of acetone in one's breath is not recommended because of the sample handling difficulty. We measured acetone in saliva by reversed-phase liquid chromatography (LC) with fluorescence (FL) detection. The proposed method was applied to the determination of acetone in the saliva of healthy volunteers and DM patients with and without ketoacidosis. Methods 3-Pentanone (I.S.) and DBD-H in acetonitrile were added to freshly collected saliva and reacted at room temperature for 20 min in the presence of trifluoroacetic acid. After the reaction, the solution was centrifuged at 10,000 ×g and 4 °C for 5 min. The supernatant was separated by reversed-phase LC and the FL detected at 550 nm (excitation at 460 nm). Results The concentrations of acetone in the DM patients with ketoacidosis were significantly higher than those of the normal subjects and DM patients without ketoacidosis. Furthermore, the total contents of the ketone bodies in the blood correlated with acetone in the saliva of the DM patients. The concentrations of acetone in the saliva of an emergency patient also correlated with the ketone bodies in the blood at each sampling time. Conclusion The proposed method using LC–FL seems to be useful for the determination of acetone in the saliva of DM patients with ketoacidosis. The method offers a new option for the diagnosis and monitoring of DM patients with ketoacidosis.

L-Pyroglutamic acid succinimidyl ester (L-PGA-OSu) and its isotopic variant (L-PGA[d5]-OSu) were newly synthesized and evaluated as the chiral labeling reagents for the enantioseparation of amino acids, in terms of separation efficiency by reversed-phase chromatography and detection sensitivity by ESI-MS/MS. The enantiomers of amino acids were easily labeled with the reagents at 60°C within 10 min in an alkaline medium containing triethylamine. Although all the diastereomers derived from 18 proteolytic amino acids could not be satisfactorily separated, the pairs of 9 amino acids were completely separated by reversed-phase chromatography using the small particle (1.7 μm) ODS column (Rs=1.95-8.05). The characteristic daughter ions, i.e., m/z 84.04 and m/z 89.04, were detected from all the derivatives by the collision induced dissociation of the protonated molecular ions. A highly sensitive detection at a low-fmol level (0.5-3.2 fmol) was also obtained from the selected reaction monitoring (SRM) chromatograms. An isotope labeling strategy using light and heavy L-PGA-OSu for the differential analysis of the DL-amino acids in different sample groups is also presented in this paper. The differential analysis of biological sample (i.e., human serum) and food product (i.e., yogurt) were tried to demonstrate the efficiency of the proposed method. The ratios of the DL-amino acids in human serum samples, spiked with the different concentrations of D-amino acids, were determined by the procedures using L-PGA-OSu and L-PGA[d5]-OSu. The D/L ratios in the two sample groups at different concentrations of amino acids were similar to the theoretical values. Furthermore, the ratios of D/L-alanine values in different yogurt products were comparable to the ratios obtained from the d/l values using only light reagent (i.e., L-PGA-OSu). Consequently, the proposed strategy is useful for the differential analysis not only in biological samples but also in food products.

The resolution of the intermediate advanced glycation end products (AGEs) in the human nail was carried out by the combination of 4,5-dimethyl-1,2-phenylenediamine (DMPD) derivatives and ultra-performance liquid chromatography with electrospray ionization time-of-flight mass spectrometry (UPLC-ESI-TOF-MS). The reaction of the reagent with 3-deoxyglucosone (3-DG), methylglyoxal (MG), and glyoxal (GO) effectively proceeds at 60°C for 2h. The resulting derivatives were efficiently separated by a gradient program (a mixture of water and acetonitrile containing 0.1% formic acid) using a reversed-phase ACQUITY UPLC BEH C(18) column (1.7 μm, 50×2.1 mm i.d.) and sensitively detected by TOF-MS. The detection limits (signal-to-noise ratio=5) of the TOF-MS were 10 to 50 fmol. A good linearity was achieved from the calibration curve, which was obtained by plotting the peak area ratios of the analytes relative to the internal standard (IS) (i.e., 2,3-hexanedione) versus the injected amounts of 3-DG, MG, and GO (r(2)>0.999), and the intra- and interday assay precisions were less than 6.89%. The derivatives of the compounds in the human nail were successfully identified by the proposed procedure. As we know, these three kinds of dicarbonyl intermediates in the formation of AGEs-3-DG, MG, and GO-were first found in human nail samples. Using these methods, the amounts of compound in the nails of healthy volunteers and diabetic patients were determined. When comparing the index from the diabetic patients with that from healthy volunteers, there is no significant difference in the content of the MG and GO in the nails. However, a statistically significant (P

The resolution of free DL-amino acids in human nail was carried out by combination of the R(-)-4-(3-isothiocyanatopyrrolidin-1-yl)-7-(N,N-dimethylaminosulfonyl)-2,1,3-benzoxadiazole [R(-)-DBD-PyNCS] derivatives and UPLC-ESI-TOF-MS. The reaction of the reagent with amino acids effectively proceeds at 55 °C for 20 min in the presence of 1% triethylamine (TEA) to produce the corresponding diastereomers. Each pair of the resulting derivatives was efficiently separated by a gradient program (a mixture of H(2)O and CH(3)CN containing 0.1% formic acid (HCOOH) or 5 mM CH(3)COONH(4) and CH(3)CN) using a reversed-phase ACQUITY UPLC™ BEH C(18) (1.7 μm, 100 mm × 2.1 mm i.d.) column and sensitively detected by TOF-MS. The detection limits (S/N=3) of the TOF-MS were 1.0-750 fmol, respectively. A good linearity was achieved from the calibration curves, which was obtained by plotting the peak area ratios of the analytes relative to the internal standard (IS), i.e., 6-aminohexanoic acid, versus the injected amounts of each amino acid (r(2)0.996), and the intra-day and inter-day assay precisions were less than 8.93%. The derivatives of the free DL-amino acids in human nail were successfully identified by the proposed procedure. As we know, for the first time, these five kinds of D-amino acids, which were D-Ala, D-Val, D-Pro, D-Ile and D-Leu, were found from human nail samples. Fifteen kinds of L-amino acids were also recognized from human nails. Using these methods, the amounts of DL-amino acids in the nails of healthy volunteers and diabetic patients were determined. When comparing the index from diabetic patients to those from healthy volunteers, there is no significant difference in the content of the L-amino acids in the nails. However, a statistically significant (P0.01) correlation was observed between the D/L-amino acid concentration ratios (Ala, Val, Ile, Leu). Therefore, because the proposed method provides a good mass accuracy and the trace detection of the DL-amino acids in human nails, this analytical technique could be a noninvasive technique to assist in the diagnosis and assessment of disease activity in diabetic patients.

A liquid chromatography/electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS) method for the determination of thyroxine (T(4)) in human saliva has been developed and validated. The saliva was deproteinized with methanol, purified using a Strata-X™ cartridge, and subjected to LC/ESI-MS/MS. Quantification was based on selected reaction monitoring, and [(13)C(6)]-T(4) was used as the internal standard. This method allowed the reproducible (intra- and inter-assay relative standard deviations

Background Polyamines are active biogenic amines which play an important role in cell growth and proliferation and the synthesis of proteins and nucleosides. In recently studies, rapid tumor growth has been associated with markedly altered polyamine biosynthesis and accumulation. Therefore, the accurate detection and identification of all the polyamines simultaneously in a single analysis is becoming more and more important for the study of their biochemical roles. Methods The development of a simultaneous determination method for 9 polyamines in human nails was attempted by the combination of nano-flow chip LC and quadrupole time-of-flight tandem mass spectrometry (Q-TOF-MS/MS). The proposed method was for the determination in the nails of healthy persons and cancer patients. Results The derivatives of the polyamines in human nail were completely separated by a gradient of an 18 min duration wash program using a reversed-phase chip column. The polyamine derivatives were then introduced into the Q-TOF-MS/MS instrument and sensitively detected in the ESI + mode. Polyamine concentration was different based on the gender, i.e., PUT and SPD is higher in men than in women in the healthy persons. Additionally, in the lung cancer patients group, as compared with the healthy persons, concentrations of PUT, N 1 -actPUT, and SPM were significantly increased. Conclusions We here present a novel sensitive, simple method for the simultaneous determination of polyamines in human nails. Furthermore, we show that polyamines (ORN, DAP, CAD, N 1 -actPUT, N 8 -actSPD, N 1 -actSPM) were detected from the human nails. Human nails may may serve as the noninvasive bio sample for diagnosis of the chronic disease.

To avoid the spreading of illegal drugs, a designated drug regulation system was introduced along with revision of the Pharmaceutical Affairs Law in Japan in 2006, and 32 substances including phenethylamine-type drugs were listed in April 2007. In this study, a new simultaneous determination method, based on ultra-fast liquid chromatography coupled with fluorescence detection (UFLC–FL), was developed for the 11 designated phenethylamine drugs. The phenethylamines were labeled with 4-(N,N-dimethylaminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (DBD-F) at 60 °C for 2 h in 0.1 M borax (pH 9.3). The resulting 11 fluorophores were completely separated by reversed-phase chromatography using an ACQUITY UPLC BEH C18 column (2.1 mm × 100 mm 1.7 μm) and fluorometrically detected at 550 nm (excitation at 450 nm). The calibration curves obtained from the peak areas versus the injection amounts of the phenethylamines showed a good linearity. The limits of detection (signal-to-noise ratio of 3: S/N = 3) on the chromatogram were in the range from 10 fmol (PMMA) to 2.5 pmol (MMDA-2). Good accuracy (%) and precision (CV) by intra-day assay and inter-day assay were also obtained using the present procedure. The method was applied to the qualitative and quantitative analyses of phenethylamine in real products obtained from the Japanese market. As the results, BDB (0.24 mg/mg), MMDA-2 (0.98 mg/mL) and 2C-I (0.016 mg/mg) were identified from the different products (powder, liquid and mushroom like). Because the procedure is simple, selective and sensitive, the present method seems to be useful for the qualitative and quantitative analyses of the designated phenethylamines in various samples including biological specimens.

The rapid, sensitive and simultaneous determination of six polyamines, i.e., ornithine (ORN), 1,3-diaminopropane (DAP), putrescine (PUT), cadaverine (CAD), spermidine (SPD) and spermine (SPM), in human hairs was performed by ultra-performance liquid chromatography (UPLC) with fluorescence (FL) detection and electrospray-ionization time-of-flight mass spectrometry (ESI-TOF-MS). The primary (-NH(2)) and secondary (-NH) amines in the polyamine structures were first labeled with 4-(N,N-dimethylaminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (DBD-F) at 60 degrees C for 30 min in the mixture of 0.1M borax (pH 9.3) and acetonitrile (CH(3)CN). The resulting derivatives were perfectly separated using an ACQUITY UPLC BEH C(18) column (1.7 microm, 100 mm x 2.1mm i.d.) by a gradient elution with a mixture of water-acetonitrile containing 0.1% formic acid (HCOOH). The separated polyamine derivatives were sensitively detected with both FL and TOF-MS. The detection limits in FL and TOF-MS were 11-86 and 2-5 fmol, respectively. However, the determination of several polyamines by FL detection was interfered with by endogenous substances in the hair. Therefore, the simultaneous determination in hair was carried out by the combination of UPLC separation and the ESI-TOF-MS detection. The structures of the polyamines were identified from the protonated-molecular ions [M+H](+) obtained from the TOF-MS measurement. A good linearity was achieved from the calibration curves, that was obtained by plotting the peak area ratios of the analytes relative to the internal standard (IS), i.e., 1,6-diaminohexane (DAH), against the injected amounts of each polyamine (0.05-50 pmol, r(2)>0.999). The proposed method was applied to the determination in the hairs of healthy volunteers. The mean concentrations of ORN, DAP, PUT, CAD, SPD and SPM in 1mg of human hairs (n=20) were 1.46, 0.18, 1.18, 0.11, 1.97 and 0.98 pmol, respectively. Because the proposed method provides a good mass accuracy and the trace detection of the polyamines in hair, this analytical technique seems to be applicable for the determination of various biological compounds in hair.

The direct determination of lipoyllysine (LLys) in proteins was carried out by reversed-phase high-performance liquid chromatography with fluorescence (FL) detection. The proteins containing alpha-lipoic acid (LA) were first hydrolyzed with several enzymes such as pronase E and subtilisin A. The disulfide bond (-S-S-) in LLys liberated from the enzyme digestion was reduced with tris(2-carboxyethyl)phosphine to the thiol form (-SH). The reduced LLys was then labeled with ammonium 4-fluoro-2,1,3-benzoxadiazole-7-sulfonate (SBD-F) at 50 degrees C for 1h. The resulting fluorophore, SBD-LLys, was separated by reversed-phase chromatography and fluorometrically detected at 510 nm (excitation at 380 nm). The calibration curve obtained from the peak areas versus the injection amounts of LLys showed a good linearity. The limits of detection and quantification of LLys on the chromatogram were approximately 0.13 pmol (signal-to-noise ratio (S/N)=3) and 0.44 pmol (S/N=10), respectively. A good recovery (98.9-107.1%) and precision (R.S.D.: 4.49-17.2%) of LLys were also obtained using the present procedure. The proposed method was used for the determination of LLys in spinach and animal tissues. The FL derivative was completely separated without any interference by endogenous substances in the sample and sensitively detected by the fluorimetry. The assay values of LLys per 1g wet tissues were 3.67 microg (kidney), 1.97 microg (liver), 2.09 microg (heart), 0.59 microg (brain), 0.30 microg (lung), 0.38 microg (pancreas), and 0.20 microg (spleen). The direct determination of LLys in protein using the FL labeling method is reported for the first time.

An exhaustive analysis of metabolites in hair samples has been performed for the first time using ultra performance liquid chromatography with electrospray ionization time-of-flight mass spectrometry (UPLC-ESI-TOF-MS). The hair samples were collected from spontaneously hypertensive model rats (SHR/Izm), stroke-prone SHR (SHRSP/Izm) and Wistar Kyoto (WKY/Izm) rats, and were analyzed by UPLC-ESI-TOF-MS; a multivariate statistical analysis method, such as the principal component analysis (PCA), was then used for screening the biomarkers. From the samples derived from the group of SHRSP/Izm at weeks 10, 18, 26 and 34, we successfully detected a potential biomarker of stroke, which existed at much higher concentrations as compared with that in the other groups. However, a significant difference could not be found at weeks less than 7 before the rats were subjected to stroke and hypertension. In addition, the present method was applicable to screening not only the disease markers, but also the markers related to aging. The method utilizing hair samples is expected to be quite useful for screening biomarkers of many other diseases, and not limited to stroke and hypertension.

Several fluorescent asparaginyl- N -acetyl- d -glucosamines (Asn-GlcNAcs), i.e., DBD-Asn-GlcNAc, NBD-Asn-GlcNAc, NDA-Asn-GlcNAc, PS-Asn-GlcNAc, FITC-Asn-GlcNAc, DMEQ-Asn-GlcNAc and DBD-PZ-Boc-Asn-GlcNAc, were synthesized as the acceptors for the resolution of oligosaccharides in glycopeptides. The resolution of oligosaccharides is based on the transglycosylation reaction with end-β- N -acetylglucosaminidase (Endo-M), isolated from Mucor hiemalis . The synthesis of each fluorescent acceptor was carried out in a one-pot reaction of Asn-GlcNAc and the corresponding fluorescent tagging reagent. The transglycosylation reaction using Endo-M proceeds at RT in neutral phosphate buffer (pH 6.0) and reached maxima at around 30 min. When Fmoc-Asn-GlcNAc (acceptor) was enzymatically reacted with Disialo-Asn (donor) in the presence of Endo-M, the ratio of Disialo-Asn-Fmoc (transglycosylation product) was almost comparable with the decreasing ratio of Fmoc-Asn-GlcNAc. Therefore, the transglycosylation activity of Endo-M from Disialo-Asn (donor) and fluorescent-Asn-GlcNAc (acceptor) was calculated from the decreasing ratio of fluorescent-Asn-GlcNAc. The order was NDA-Asn-GlcNAc > DBD-Asn-GlcNAc ≅ Fmoc-Asn-GlcNAc > NBD-Asn-GlcNAc ≅ DMEQ-Asn-GlcNAc > DNS-Asn-GlcNAc > PS-Asn-GlcNAc > FITC-Asn-GlcNAc. On the other hand, the activity with a fluorescent acceptor (DBD-PZ-Boc-Asn-GlcNAc), labeled to a carboxylic acid group in the Asn residue, was the strongest among the synthesized acceptors.