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Riociguat is novel antihypertensive drug for treatment of pulmonary hypertension. As such, it is still being tested in many clinical and pharmacokinetic trials. Existing methods that determine serum riociguat and desmethylriociguat (DMR) are based solely on liquid chromatography with mass spectrometry. Therefore, we present a novel capillary electrophoresis with mass spectrometry method (CE-MS) for their determination in human serum as alternative method for ongoing trials. Complete resolution of both analytes was achieved by means of pH optimization of ammonium formate background electrolytes, that are fully compatible with ESI/MS detection. Simple liquid-liquid extraction was used as sample pretreatment. The calibration dependence of the method was linear (in the range of 10 - 1000 ng/mL), with adequate accuracy (90.1-114.9%) and precision (13.4%). LOD and LOQ were arbitrarily set at 10 ng/mL for both analytes. Clinical applicability was validated using serum samples from patients treated with riociguat in pharmacokinetic study and the results corresponded with reference HPLC-MS/MS values. Capillary electrophoresis proved to be sensitive and selective tool for the analysis of riociguat and DMR. This article is protected by copyright. All rights reserved.

Perampanel is a novel antiepileptic drug used in paediatric patients. Existing methods that determine serum perampanel are of limited practicability. We developed a novel capillary electrophoresis (CE) method using a new version of acetonitrile stacking for on-line sample pre-concentration, and fluorescence detection (FD). CE separations were performed in a fused-silica capillary where the electroosmotic flow was reduced by coating the inner surface using a INST coating solution. The optimised background electrolyte composition was 50 mM chloroacetic acid with addition of 0.5% m/v polyvinylalcohol (pH 2.15) and separation was driven by application of positive voltage + 30 kV. Serum samples (25 μL) treated by the addition of acetonitrile in a ratio of 1:3 v/v were each injected into the capillary at a large volume that corresponded to the length (129 mm) of the sample zone (hydrodynamic pressure impulse 6000 mbars). Acetonitrile stacking is based on the forcing the sample zone out of the capillary with simultaneous application of the separation voltage. Under such conditions, the enhancing factor achieves the value 57 for peak area compared to the small sample injection length (3.2 mm, hydrodynamic pressure impulse 150 mbar.s). A fluorescence detector with a broad excitation filter (240–400 nm) and an emission filter (495 nm) was used for visualisation of the native fluorescence of perampanel. The calibration dependence of the method was linear (in the range of 10–1000 ng mL−1), with adequate accuracy (99.8–103.3 %) and precision (13.1%). LOD and LOQ for perampanel were 2.9 ng mL-1 and 9.5 ng mL−1, respectively. Clinical applicability was validated using serum samples from patients treated with perampanel and the results corresponded with reference LC–MS/MS values. Our method offers a promising alternative for determining serum perampanel with several advantages. In particular, the low quantity of serum (25 μL) required means that testing can be performed on samples obtained for monitoring other antiepileptic medications, and thus reduces the test-burden on paediatric patients.

Riociguat is a novel antihypertensive drug for the treatment of pulmonary hypertension. We present electrophoretic characterization, i.e. migration behavior of riociguat and metabolite M1 as support for optimized CZE/MS assay. Fundamental separation parameters, such as peak width, symmetry, and resolution are studied in a series of ammonium formate buffers within pH range 2.60-5.61. The narrow region of peak symmetry lies close to pH 4.0 for both analytes. Accordingly, the value of resolution maximizes in a background electrolyte adjusted to pH 4.10. Basic calibration parameters estimated from CZE experiments with absorption photometric and mass spectrometric detection of riociguat and metabolite M1 were evaluated. More than three orders lower LOD was achieved with high resolution mass spectrometric detection. The observed difference in the sensitivity of both detection techniques gives priority to the utilization of CZE/MS in practice. The values of dissociation constants of riociguat and metabolite M1, pKBH , were determined from CZE measurements in lithium formate and lithium acetate background electrolytes with constant ionic strength. The value of pKBH = 4.30 ± 0.02 for riociguat corresponds well to the value already presented in the literature. According to our observation, metabolite M1 behaves like a slightly stronger base with estimated pKBH = 4.40 ± 0.02.

In the last decade there has been a dramatic increase in the availability and abuse of synthetic cathinones – new amphetamine-like stimulants. Even though their abuse during pregnancy could have serious adverse effects on the fetus, cathinones are not readily included in neonatal toxicological screenings. Meconium (first neonatal stool) is the specimen of choice to reveal long term drug exposure, however as it is a highly complex matrix, the sample preparation is a critical step before the instrumental analysis. The aim of this work was to develop a suitable meconium sample extraction technique using the advantages of salting-out assisted liquid-liquid extraction (SALLE) and using only MS-friendly organic ammonium salts. We further developed and validated liquid chromatography tandem-mass spectrometry method for the determination of ‘traditional’ stimulants (methamphetamine, amphetamine, MDMA) and cathinones (mephedrone, methylenedioxypyrovalerone (MDPV), α-pyrrolidinopentiophenone (α-PVP), methylone, butylone, flephedrone, and naphyrone). Matrix-matched calibration was prepared in the concentration range 10–2000 ng/g. The limits of quantification were determined as 10 ng/g, recoveries ranged from 48.2% to 94.3% and the matrix effect was between 60.2% and 101.4%. Accuracy (86.1–114.5%) and precision (4.9–14.9%) were determined and all validation criteria were met for all analytes except for naphyrone. Finally, our analytical method was tested on a set of real meconium samples, which were found positive for amphetamine, methamphetamine and methylone, thus demonstrating the validity of the method.

High anion gap metabolic acidosis frequently complicates acute paracetamol overdose and is generally attributed to lactic acidosis or compromised hepatic function. However, metabolic acidosis can also be caused by organic acid 5-oxoproline (pyroglutamic acid). Paracetamol's toxic intermediate, N-acetyl-p-benzoquinoneimine irreversibly binds to glutathione and its depletion leads to subsequent disruption of the gamma glutamyl cycle and an excessive 5-oxoproline generation. This is undoubtedly an underdiagnosed condition because measurement of serum 5-oxoproline level is not readily available. A simple, cost effective, and fast capillary electrophoresis method with diode array detection (DAD) for simultaneous measurement of both paracetamol (acetaminophen) and 5-oxoproline in serum was developed and validated. This method is highly suitable for clinical toxicology laboratory diagnostic, allowing rapid quantification of acidosis inducing organic acid 5-oxoproline present in cases of paracetamol overdose. The calibration dependence of the method was proved to be linear in the range of 1.3-250μgmL-1, with adequate accuracy (96.4-107.8%) and precision (12.3%). LOQ equaled 1.3μgmL-1 for paracetamol and 4.9μgmL-1 for 5-oxoproline.

A simple, cost effective, and fast gas chromatography method with mass spectrometry detection (GC-MS) for simultaneous measurement of formic acid, glycolic acid, methoxyacetic acid, ethoxyacetic acid and 2-hydroxyethoxyacetic acid in serum and urine was developed and validated. This multi-analyte method is highly suitable for clinical and emergency toxicology laboratory diagnostic, allowing identification and quantification of five most common acidosis inducing organic acids present in cases of alcohol intoxication. Furthermore, when patients are admitted to emergency unit at late stage of toxic alcohol intoxication, the concentration of parent compound may be already low or not detectable. This new method employs a relatively less used class of derivatization agents - alkyl chloroformates, allowing the efficient and rapid derivatization of carboxylic acids within seconds. The entire sample preparation procedure is completed within 5 min. The optimal conditions of derivatization procedure have been found using chemometric approach (design of experiment). The calibration dependence of the method was proved to be quadratic in the range of 25-3000 mg L(-1), with adequate accuracy (97.3-108.0%) and precision (12.8%). The method was successfully applied for identification and quantification of the selected compounds in serum of patients from emergency units.

Objectives A simple, cost-effective, and fast gas chromatography method with mass spectrometry detection (GC-MS) for simultaneous measurement of ethylene glycol, 1,2-propylene glycol and glycolic acid was developed and validated for clinical toxicology purposes. Design and methods Successful derivatization of glycolic acid with isobutyl chloroformate was achieved directly in serum/urine while glycols are simultaneously derivatized by phenylboronic acid. The entire sample preparation procedure is completed within 10 min. Results The assay was proved to be quadratic in the range of 50 to 5000 mg L − 1 with adequate accuracy (96.3–105.8%) and precision (CV ≤ 8.9%). Conclusion The method was successfully applied to quantify the selected compounds in serum of patients from emergency units and the results correlated well with parallel GC-FID measurements ( R 2 0.9933 for ethylene glycol and 0.9943 for glycolic acid).

Objectives The aim of this study was to develop and validate a gas chromatographic method with flame ionization detection (GC–FID) for the measurement of ibuprofen, naproxen and ketoprofen for clinical toxicology purposes. Design and methods 100 μL of plasma was treated with methyl chloroformate and derivatized analytes were extracted with hexane. Optimal conditions of the derivatization procedure have been found using the experimental chemometric design (face-centered central composite design). The selectivity and efficiency of the procedure was confirmed by GC–MS. Results The assay was linear in the concentration range of 10–400 μg mL − 1 , with adequate accuracy and precision for GC–FID (98–106.7%, CV ≤ 9.1%, respectively) and for GC–MS (99.3–105.5%, CV ≤ 9.2%, respectively). Conclusion The entire sample preparation procedure is completed within 5 min and the quantitative results are available within 35 min. The method was successfully applied to quantify the selected compounds in serum of patients from emergency units.