Acid In Human Plasma Research Articles

First identification of dimethoxycinnamic acids in human plasma after coffee intake by liquid chromatography–mass spectrometry

There is a substantial amount of published literature on the bioavailability of various coffee components including the most abundant metabolites, caffeic and ferulic acids. Surprisingly, to date, the appearance of dimethoxycinnamic acid derivatives in humans has not been reported despite the fact that methylated form of catechol-type polyphenols could help maintain, modify or even improve their biological activities. This study reports an LC–MS method for the detection of dimethoxycinnamic acid in human plasma after treatment with an esterase. Liquid chromatography, including the combination of methanol and acetonitrile as organic eluent, was optimized to resolve all interferences and enable reliable detection and identification of 3,4-dimethoxycinnamic and 3,4-dimethoxy-dihydrocinnamic acids. In addition to the good mass accuracy achieved (better than 5ppm), tandem mass spectrometric and co-chromatography experiments further confirmed the identity of the compounds. The optimized method was applied to analyze samples obtained immediately, 1 and 10h after coffee ingestion. The results show that in particular 3,4-dimethoxycinnamic acid appears in high abundance (∼380nM at 60min) in plasma upon coffee intake, indicating that it is important to consider these derivatives in future bioavailability and bioefficacy studies.

Analisis Asam Valproat dalam Plasma Secara Kromatografi Gas

Valproic acid is an anticonvulsant drug that works by increasing the levels of γ-aminobutyric acid (GABA). Determination of valproic acid is quite difficult because it has no chromophore groups in its structure. Aa analytical method using gas chromatography (GC) with flame ionization detector for the determination of valproic acid in human plasma has been developed and optimized. Valproic acid was extracted from plasma by liquid-liquid extraction method using diethyl ether. The optimum analysis conditions for valproic acid in plasma were achieved by regulated gas chromatography injector and detector at a temperature of 250oC and temperature programming with an initial temperature of 70oC and 5oC temperature increasing per minute until a temperature of 100oC, then held for 1 minute. Then the temperature was increased by 2°C per minute until the column temperature to 150oC. The optimum conditions of analysis took 32 minutes. In the concentration range from 40.0 to 100.0 µg/mL yielded a linear calibration curve with correlation coefficient (r) of 0.9894. Accuracy (% diff) of this method was -13.67% to 12.33% with precision (CV) between 9.33% to 14.92%, and relative recovery test was 86.33% to 112.33%. Keywords : gas chromatography, optimation, plasma in vitro, valproic acid

LC–MS–MS Quantitative Determination of Ursolic Acid in Human Plasma and Its Application to Pharmacokinetic Studies

A sensitive and specific liquid chromatography–electrospray ionization–tandem mass spectrometry method has been developed and validated for the identification and quantification of brivudine in human plasma using diclofenac as an internal standard. The method involves extraction with ethyl acetate. The analyte was separated on a C18 column and analyzed in multiple reaction monitoring mode with a negative electrospray ionization interface using the [M–H]− ions, m/z 332.8→m/z 80.9 for brivudine, m/z 293.6→m/z 249.5 for diclofenac. The method was validated over the concentration range of 5.54–2,836 μg L−1 for brivudine. The intra-and inter-day precisions were less than 8.91% in terms of relative standard deviation (RSD), and the accuracy was within −4.22% in terms of relative error (RE). The lower limit of quantification (LLOQ) was 5.54 μg L−1 with acceptable precision and accuracy. There were almost no matrix effects. Recovery of brivudine spiked in drug-free plasma was higher than 77.17%. The method was used to study the pharmacokinetic profile of brivudine in human plasma after oral administration of brivudine tablets.

Development and validation of a sensitive and selective UHPLC–MS/MS method for simultaneous determination of both free and total eicosapentaeonic acid and docosahexenoic acid in human plasma

A sensitive, selective, and quantitative method for the simultaneous determination of free and total eicosapentaeonic acid (EPA) and docosahexenoic acid (DHA) has been developed and validated in human plasma using fatty acid free human serum albumin as a surrogate matrix. Clean-up for free EPA and DHA employs a liquid–liquid extraction with hexane to remove plasma interferences and provide for cleaner chromatography. The method for total EPA and DHA requires a digestion of the triglycerides followed by liquid–liquid extraction with hexane. Ultra high performance liquid chromatography (UHPLC) technology on a BEH C18 stationary phase column with 1.7 μm particle size was used for chromatographic separation, coupled to tandem mass spectrometry (UHPLC–MS/MS). The method for free EPA and DHA was validated over the concentration range of 0.05–25 μg/mL, while total EPA and DHA concentration range was 0.5–250 μg/mL. The results from assay validation show that the method is rugged, precise, accurate, and well suited to support pharmacokinetic studies. To our knowledge, this work represents the first UHPLC–MS/MS based method that combines both free and total EPA and DHA with a relatively small sample volume (25 μL aliquot) and a run time of 1.5 min, facilitating automation and high throughput analysis.

HPLC Method for Quantification of Oxidative Stress by Salicilate Hydroxylation in Human Plasma

The aim of the present study was to modify and validate a high-performance liquid chromatographic (HPLC) method for determining 2,3 and 2,5 dihydroxybenzoic acid (2,3-DHBA and 2,5-DHBA) from salicylic acid in human plasma. The mobile phase was a mixture of sodium acetate/citrate (pH 2.5) 30 mM-methanol (93:7, v/v). The injection volume was 10 muL. Retention time for 2,5-DHBA, and 2,3-DHBA was 4.5 +/- 0.10 and 5.8 +/- 0.15 min, respectively. The detection and quantification limits were 10 and 40 nM for 2,3-DHBA and 8 and 20 nM for 2,5-DHBA. Linearity was evaluated in the range of 40-1600 nM for both metabolites. Inter- and intra-analysis variation coefficient was below 10%. Good recoveries of more than 99% were obtained for both metabolites using this method.

Determination of lipoic acid in human plasma by HPLC-ECD using liquid–liquid and solid-phase extraction: Method development, validation and optimization of experimental parameters

A rapid, inexpensive, sensitive and specific HPLC-ECD method for the determination of lipoic acid in human plasma was developed and validated over the linearity range of 0.001–10 μg/ml using naproxen sodium as an internal standard (IS). Extraction of lipoic acid and IS from plasma (250 μl) was carried out with a simple one step liquid–liquid extraction using dichloromethane. Similarly solid-phase extraction was carried out using dichloromethane as extraction solvent. The separated organic layer was dried under the stream of nitrogen at 40 °C and the residue was reconstituted with the mobile phase. Complete separation of both lipoic acid and IS at 30 °C on Discovery HS C18 RP column (250 mm × 4.6 mm, 5 μm) was achieved in 6 min using 0.05 M phosphate buffer (pH 2.5 adjusted with phosphoric acid):acetonitrile (50:50, v/v) as a mobile phase pumped at the rate of 1.5 ml/min using electrochemical detector in DC mode at the detector potential of 1.0 V. The limit of detection and limit of quantification of lipoic acid were 200 pg/ml and 1 ng/ml, respectively. While on column limit of detection and limit of quantification of lipoic acid were 10 and 50 pg/ml, respectively. The absolute recoveries of lipoic acid with liquid–liquid and solid-phase extraction were 98.43, 95.65, 101.45, and 97.36, 102.73, 100.17% at 0.5, 1 and 5 μg/ml levels, respectively. Coefficient of variations for both intra-day and inter-day were between 0.28 and 4.97%. The method is validated and will be quite suitable for the analysis of lipoic acid in the plasma of human volunteers as well as patients with diabetes and cardiovascular diseases.

Determination of plasma concentration of cinnamic acid by LC-MS-MS and study of pharmacokinetics in healthy volunteers after Mailuoning injection

To establish a LC-MS-MS method for quantification of cinnamic acid in human plasma and study its pharmacokinetics after administration of Mailuoning injection at a single and the multiple doses to healthy volunteers. Plasma samples were acidified with hydrochloric acid and extracted with ethyl acetate-ether. Cinnamic acid was determined by LC-MS-MS using a ZOBAX SB C18 column with a mobile phase of methanol-water (containing 2 mmol x L(-1) ammonium acetic) (45: 55) at a flow rate of 0.5 mL x min(-1) and detected using ESI with negative ionization mode. Ions monitored in the multiple reaction monitoring (MRM) mode were m/z 146.8 /103.1 [M - H]- for cinnamic acid and m/z 245.6 /126.0 [M - H] - for tinidazole (IS). After administration of Mailuoning injection at a single and the multiple doses via intravenous guttae (ivgtt) to 10 healthy volunteers, the concentration of cinnamic acid in plasma was determined by LC-MS-MS method. The concentration-time curves were simulated by DASver 1.0 and the pharmacokinetic parameters were calculated. The calibration curve was linear within the range of 0.5400 microg x L(-1). The LLOQ was 0.5 microg x L(-1) and RSDs of intra and inter day were less than 10%. The concentration-time curves of cinnamic acid were consistent with the two-compartment model. The pharmacokinetic parameters after administration of Mailuoning Injection at a single and the multiple injections were as follows: C(max) (microg x L(-1)), 115.73 +/- 44.31 and 113.79 +/- 25.61; T1/2beta (h), 0.41 +/- 0.087 and 0.52 +/- 0.132; V(L x kg(-1)), 0.519 +/- 0.134 and 0.651 +/- 0.322; CL(L x kg(-1) x h(-1)), 0.899 +/- 0.295 and 0.830 +/- 0.222; AUC(0-tn) (microg x L(-1) x h), 158.64 +/- 56.019 and 166.49 +/- 46.788. The developed LC-MS/MS method was sensitive and selective, and there was no interference from endogenous substances. After administration of Mailuoning injection via ivgtt to healthy volunteers, the pharmacokinetic parameters of cinnamic acid between a single and the multiple doses, and between the male and female were no significant difference. There was no accumulation with multiple injections for cinnamic acid, but there were significant individual differences in the pharmacokinetics of cinnamic acid in volunteers.

LC-MS–MS Simultaneous Determination of Niacin, Niacinamide and Nicotinuric Acid in Human Plasma LC-MS–MS and Its Application to a Human Pharmacokinetic Study

A highly selective and sensitive liquid chromatographic tandem mass spectrometric (LC-MS–MS) method was developed and validated for the quantitation and pharmacokinetic study of niacin (NA) and its two metabolites niacinamide (NAM) and nicotinuric acid (NUR) in human plasma. Protein precipitation with 14% perchloric acid solution was selected for sample preparation, and ganciclovir was used as an internal standard. Separation was on a Phenomenex Curosil-PFP (250 mm × 4.6 mm, 5 μm) column by a multiple steep steps linear gradient elution with mobile phase consisting of water and methanol, both containing 0.1% formic acid, pumped at a flow rate of 1 mL min−1. The determination was optimized and carried out with positive electrospray ionization by selective multiple reaction monitoring. The method was linear in the concentration range of 15–2,000 ng mL−1 for NA, 70–2,000 ng mL−1 for NAM and 10–2,000 ng mL−1 for NUR, by standard addition calibration. The application of LC-MS–MS was demonstrated for the specific and quantitative analysis of NA, NAM and NUR in human plasma from a pharmacokinetic study in 12 healthy Chinese volunteers treated with three incremental doses of niacin extended-release/lovastatin tablets and an additional steady-state regime.

Rapid quantification of conjugated and unconjugated bile acids and C27 precursors in dried blood spots and small volumes of serum

The aim of the study was to develop a method for fast and reliable diagnosis of peroxisomal diseases and to facilitate differential diagnosis of cholestatic hepatopathy. For the quantification of bile acids and their conjugates as well as C(27) precursors di- and trihydroxycholestanoic acid (DHCA, THCA), in small pediatric blood samples we combined HPLC separation on a reverse-phase C18 column with ESI-MS/MS analysis in the negative ion mode. Analysis was done with good precision (CV 3,7%-11.1%) and sufficient sensitivity (LOQ: 11-91 nmol/L) without derivatization. Complete analysis of 17 free and conjugated bile acids from dried blood spots and 10 microL serum samples, respectively, was performed within 12 min. Measurement of conjugated primary bile acids plus DHCA and THCA as well as ursodeoxycholic acid was done in 4.5 min. In blood spots of healthy newborns, conjugated primary bile acids were found in the range of 0.01 to 2.01 micromol/L. Concentrations of C(27) precursors were below the detection limit in normal controls. DHCA and THCA were specifically elevated in cases of peroxysomal defects and one Zellweger patient.

Spectrophotometric determination of carminic acid in human plasma and fruit juices by second order calibration of the absorbance spectra–pH data matrices coupled with standard addition method

A simple analytical method based on the second-order calibration of the pH gradient spectrophotometric data was developed for assay of carminic acid (CA) in human plasma and orange juice over the concentration range of 1.5–14.0 μM. The multi-way data analysis method was coupled with standard addition to encounter the significant effects of plasma and juices matrices on the acid–base behavior and UV–vis. absorbance spectra of CA. Thus, the standard addition three-way calibration data of plasma or fruit juices samples were analyzed by parallel factor analysis (PARAFAC) and the concentration related scores were used to derive a standard addition plot such as one obtained in univariate standard addition method. The number of PARAFAC components was obtained utilizing different criteria such as core consistency and residual errors through pf-test implementation. The applicability of the proposed method was evaluated by analysis of human plasma and fruit juices spiked with different levels of standard CA solutions. The results confirmed the success of the proposed method in the analysis of pH gradient spectrophotometric data for determination of CA. The recoveries were between 86.7 and 106.7.

Development of a Hydrophilic Liquid Interaction Chromatography−High-Performance Liquid Chromatography−Tandem Mass Spectrometry Based Stable Isotope Dilution Analysis and Pharmacokinetic Studies on Bioactive Pyridines in Human Plasma and Urine after Coffee Consumption

The paper reports on the development of an accurate hydrophilic liquid interaction chromatography tandem mass spectrometry (HILIC-MS/MS) based stable isotope dilution analysis for the simultaneous quantitation of the food-derived bioactive pyridines trigonelline, nicotinic acid, nicotinamide, and N-methylpyridinium, as well as their key metabolites nicotinamide-N-oxide, N-methylnicotinamide, N-methyl-2-pyridone-5-carboxamide, N-methyl-4-pyridone-5-carboxamide, and N-methyl-2-pyridone-5-carboxylic acid in human plasma and urine. Precision of the stable isotope dilution analysis (SIDA) was 1.9% and 11.9% relative standard deviation (n = 6), and accuracy was between 92.4% and 113.0%. The lower limit of quantitation (LLOQ) was 50 fmol (10 pmol/mL) injected onto the column for all analytes with the exception of N-methyl-2-pyridone-5-carboxylic acid and N-methyl-2-pyridone-5-carboxamide, for which an LLOQ of 100 fmol (20 pmol/mL) was found. The method was applied to monitor the plasma appearance and urinary excretion and to determine pharmacokinetic parameters of the bioactive pyridines as well as their metabolites in a clinical human intervention study with healthy volunteers (six women, seven men) after oral administration of 350 mL of a standard coffee beverage. Trigonelline plasma levels increased from 160 nmol/L to maximum concentrations of 5479 (males) or 6547 nmol/L (females), and N-methylpyridinium plasma levels raised from virtually complete absence to maximum values of 777 (females) or 804 nmol/L (males) within 2-3 and 1-2 h after coffee consumption, respectively. The high plasma levels of N-methylpyridinium found after coffee consumption clearly demonstrate for the first time that this cation is entering the vascular system, which is the prerequisite for biological in vivo effects claimed for that compound. In contrast, the coffee intervention did not significantly influence the plasma concentrations of N-methyl-2-pyridone-5-carboxamide and N-methyl-4-pyridone-5-carboxamide, the major niacin metabolites. Within 8 h after coffee intervention, an urinary excretion of 57.4 +/- 6.9% of trigonelline and 69.1 +/- 6.2% of N-methylpyridinium was found for the male volunteers, whereas females excreted slightly less with 46.2 +/- 7.4% and 61.9 +/- 12.2% of these pyridines.

High-throughput salting-out assisted liquid/liquid extraction with acetonitrile for the simultaneous determination of simvastatin and simvastatin acid in human plasma with liquid chromatography

Simvastatin (SS) is an effective cholesterol-lowering medicine, and is hydrolyzed to simvastatin acid (SSA) after oral administration. Due to SS and SSA inter-conversion and its pH and temperature dependence, SS and SSA quantitation is analytically challenging. Here we report a high-throughput salting-out assisted liquid/liquid extraction (SALLE) method with acetonitrile and mass spectrometry compatible salts for simultaneous LC–MS/MS analysis of SS and SSA. The sample preparation of a 96-well plate using SALLE was completed within 20 min, and the SALLE extract was diluted and injected into an LC–MS/MS system with a cycle time of 2.0 min/sample. The seamless interface of SALLE and LC–MS eliminated drying down step and thus potential sample exposure to room or higher temperature. The stability of SS and SSA in various concentration ratios in plasma was evaluated at room and low (4 °C) temperature and the low temperature (4 °C) was found necessary to maintain sample integrity. The short sample preparation time along with controlled temperature (2–4 °C) and acidity (pH 4.5) throughout sample preparation minimized the conversion of SS → SSA to ≤0.10% and the conversion of SSA → SS to 0.00% The method was validated with a lower limit of quantitation (LLOQ) of 0.094 ng mL −1 for both SS and SSA and a sample volume of 100 μL. The method was used for a bioequivalence study with 4048 samples. Incurred sample reproducibility (ISR) analysis of 362 samples from the study exceeded ISR requirement with 99% re-analysis results within 100 ± 20% of the original analysis results.

HIGH PERFORMANCE LIQUID CHROMATOGRAPHIC ESTIMATION OF DROTAVERINE HYDROCHLORIDE AND MEFENAMIC ACID IN HUMAN PLASMA

The present work describes a reverse phase HPLC method for the quantitation of drotaverine hydrochloride and mefenamic acid in human plasma. Organic solvent system used for liquid extraction composed of dichloromethane, and isopropyl alcohol in the ratio 80:20 (v/v). The compounds were separated on a Thermo BDS Hypersil C 8 (25.0 cm × 4.6 mm, 5 µm particle size) column in isocratic mode with a mixture of acetonitrile and ammonium acetate buffer (20 mM, pH 3.5 ± 0.05 adjusted with 85% phosphoric acid) in a ratio of 55: 45 (v/v), as the mobile phase, at a flow rate of 1.0 mL min -1 . The effluent was monitored by UV detection at 230 nm. The method was validated for accuracy, precision, specificity, linearity, and sensitivity. The total chromatographic analysis time was approximately 10 min with diclofenac sodium (internal standard), drotaverine hydrochloride and mefenamic acid, eluting with retention times of 6.89, 8.21 and 9.67 min, respectively. Calibration plots were linear within the range of 32-960 ng mL -1 and 100-3000 ng mL -1 for drotaverine hydrochloride and mefenamic acid, respectively. The limit of detection values were 11 and 33 ng mL -1 and the limit of quantitation values were 32 and 100 ng mL -1 for drotaverine hydrochloride and mefenamic acid, respectively. Results from analysis of quality control samples at concentrations of 90, 450 and 750 ng mL -1 for drotaverine hydrochloride and 300, 1600 and 2400 ng mL -1 for mefenamic acid, were indicative of good repeatability and precision. Recovery from plasma was 98.68% for drotaverine hydrochloride and 94.51% for mefenamic acid.

High performance liquid chromatography–tandem mass spectrometry for the determination of bile acid concentrations in human plasma

We report a sensitive and robust method to determine cholic acid (CA), chenodeoxycholic acid (CDCA), deoxycholic acid (DCA), lithocholic acid (LCA), ursodeoxycholic acid (UDCA), and their taurine- and glycine-conjugate concentrations in human plasma using liquid chromatography–tandem mass spectrometry. Activated charcoal was utilized to prepare bile acid-free plasma, which served as the biological matrix for the preparation of standard and quality control samples. Plasma sample preparation involved solid-phase extraction. A total of 16 bile acids and 5 internal standards were separated on a reverse column by gradient elution and detected by tandem mass spectrometry in negative ion mode. The calibration curve was linear for all the bile acids over a range of 0.005–5 μmol/L. The extraction recoveries for all the analytes fell in the range of 88–101%. Intra-day and inter-day coefficients of variation were all below 10%. A stability test showed that all the bile acids were stable in plasma for at least 6 h at room temperature, at least three freeze–thaw cycles, in the −70 °C or −20 °C freezer for 2 months, and also in the reconstitution solution at 8 °C for 48 h. Comparison of the matrix effect of bile acid-free plasma with that of real plasma indicated that the charcoal purification procedure did not affect the properties of charcoal-purified plasma as calibration matrix. This method has been used to determine the bile acid concentrations in more than 300 plasma samples from healthy individuals. In conclusion, this method is suitable for the simultaneous quantification of individual bile acids in human plasma.

Liquid chromatograph/tandem mass spectrometry assay for the simultaneous determination of chlorogenic acid and cinnamic acid in plasma and its application to a pharmacokinetic study

A rapid and high sensitive liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for simultaneous determination of chlorogenic acid and cinnamic acid in human plasma was developed. The analytes and internal standard (IS), tinidazole, were extracted from human plasma via liquid/liquid extraction with ether–ethyl acetate (1:1, v/v) and separated on an Agilent Zorbax SB C18 column within 5 min. Quantitation was performed on a triple quadrupole mass spectrometer employing electrospray ionization technique, operating in multiple reaction monitoring (MRM) and negative ion mode. The precursor to product ion transitions monitored for chlorogenic acid, cinnamic acid and IS were m/ z 352.9 → 191.1, 146.8 → 103.1, 245.6 → 126.0, respectively. The assay was validated with linear range of 1.00–800.00 ng/mL for chlorogenic acid and 0.50–400.00 ng/mL for cinnamic acid. The intra- and inter-day precisions (RSD%) were within 9.05% for each analyte. The absolution recoveries were greater than 74.62% for chlorogenic acid and 76.21% for cinnamic acid. Each analyte was proved to be stable during all sample storage, preparation and analytic procedures. The method was successfully applied to a pharmacokinetic study of Mailuoning injection in 10 healthy volunteers.

Rapid determination of losartan and losartan acid in human plasma by multiplexed LC–MS/MS

A rapid LC-MS/MS method has been developed and validated for the determination of losartan (LOS) and its metabolite losartan acid (LA) (EXP-3174) in human plasma using multiplexing technique (two HPLC units connected to one MS/MS). LOS and LA were extracted from human plasma by SPE technique using Oasis HLB cartridge without evaporation and reconstitution steps. Hydroflumethiazide (HFTZ) was used as an internal standard (IS). The analytes were separated on Zorbax SB C-18 column. The mass transition [M-H] ions used for detection were m/z 421.0 --> 127.0 for LOS, m/z 435.0 --> 157.0 for LA, and m/z 330.0 --> 239.0 for HFTZ. The proposed method was validated over the concentration range of 2.5-2000 ng/mL for LOS and 5.0-3000 ng/mL for LA with correlation coefficient > or = 0.9993. The overall recoveries for LOS, LA, and IS were 96.53, 99.86, and 94.16%, respectively. Total MS run time was 2.0 min/sample. The validated method has been successfully used to analyze human plasma samples for applications in 100 mg fasted and fed pharmacokinetic studies.

Nitro‐fatty Acids Occur in Human Plasma in the Picomolar Range: a Targeted Nitro‐lipidomics GC–MS/MS Study

First studies on the occurrence of nitrated fatty acids in plasma of healthy subjects revealed basal concentrations of 600 nM for free/nonesterified nitro-oleic acid (NO(2)-OA) as measured by liquid chromatography tandem mass spectrometry (LC-MS/MS). We recently showed by a gas chromatography tandem mass spectrometry (GC-MS/MS) method the physiological occurrence of two isomers, i.e., 9-NO(2)-OA and 10-NO(2)-OA, at mean basal plasma concentrations of 880 and 940 pM, respectively. In consideration of this large discrepancy we modified our originally reported method by replacing solid-phase extraction (SPE) by solvent extraction with ethyl acetate and by omitting the high-performance liquid chromatography (HPLC) step for a more direct detection and with the potential for lipidomics studies. Intra-assay imprecision and accuracy of the modified method in human plasma were 1-34% and 91-221%, respectively, for added NO(2)-OA concentrations in the range 0-3,000 pM. This method provided basal plasma concentrations of 306 +/- 44 pM for 9-NO(2)-OA and 316 +/- 33 pM for 10-NO(2)-OA in 15 healthy subjects. Nitro-arachidonic acid and nitro-linolenic acid were not detectable in the plasma samples. In summary, our studies show 9-NO(2)-OA and 10-NO(2)-OA as endogenous nitrated fatty acids in human plasma in the pM range; HPLC is recommendable as a sample clean-up step for reliable quantification of nitro-oleic acids by GC-MS/MS.

Determination of arotinoid acid in human plasma by liquid chromatography–tandem mass spectrometry

Arotinoid acid (Ro 13-7410) is the third generation of synthetic retinoid, which was developed for the treatment of psoriasis and other hyperkeratotic skin disorders. The therapeutically active dose is less than 0.5 μg/kg body weight/day. To investigate the pharmacokinetics of arotinoid acid, a sensitive and selective liquid chromatographic–tandem mass spectrometric method (LC–MS/MS) for the determination of arotinoid acid in human plasma was developed and validated. The sample processing was carried out in the dark to minimize photodegradation of the analytes. Arotinoid acid and the internal standard (IS), acitretin, were extracted from plasma samples using solid phase extraction with Oasis HLB cartridges. Chromatographic separation was achieved on a Zorbax Extend C 18 column (150 mm × 4.6 mm, i.d., 5 μm) using methanol:acetonitrile:5 mM ammonium acetate (48:32:20, v/v/v) as the mobile phase at a flow rate of 0.8 mL/min. The detection was carried out in multiple reaction monitoring (MRM) mode via negative electrospray ionization (ESI) interface. The lower limit of quantification (LLOQ) achieved was 37.1 pg/mL with intra-day and inter-day precision (RSD) of 8.7% and 8.5%, and accuracy (RE) of 2.0%. Inter-day and intra-day RSD for three quality control levels (QCs) across validation runs were less than 11.0% and the accuracy ranged from 1.9% to 3.3%. The validated LC–MS/MS method was applied to a phase I clinical pharmacokinetic study after a single oral administration of 40 μg arotinoid trometamol to healthy subjects.

N-(1-Naphthyl)ethylenediamine, a New UV Labeling Reagent Used for LC Determination of Valproic Acid in Human Plasma

A high performance liquid chromatography method is presented for the determination of valproic acid levels in human plasma. The method was based on pre-column derivatization using N-(1-naphthyl)ethylenediamine as a new labeling agent. The calibration curve was linear in the investigated concentration range between 0.1 and 100 μg mL−1 and showed good accuracy and reproducibility. The assay provided a limit of quantification of 0.1 μg mL−1 for valproic acid and a limit of detection of 10 ng mL−1, respectively. The presented method was successfully applied to the determination of valproic acid levels in plasma after oral administration of 600 or 800 mg of sodium valproate.

Determination of fenofibric acid in human plasma by ultra performance liquid chromatography–electrospray ionization mass spectrometry: application to a bioequivalence study

A rapid, specific and sensitive ultra-performance liquid chromatography tandem mass spectrometry method was developed for the determination of fenofibric acid in human plasma. The method involves simple, one-step liquid-liquid extraction procedure coupled with an Acquity UPLC(TM) BEH C(18) column (50 x 2.1 mm, i.d., 1.7 microm) with isocratic elution at a flow-rate of 0.2 mL/min and mefenamic acid was used as the internal standard. The Quattro Premier XE mass spectrometry was operated under the multiple reaction-monitoring mode using the electrospray ionization technique. Using 250 microL plasma, the methods were validated over the concentration rang 0.05-7.129 microg/mL, with a lower limit of quantification of 0.05 microg/mL. The intra- and inter-day precision and accuracy were within 9.3%. The recovery was 66.7% and 52.6% for fenofibric acid, and mefenamic acid, respectively. Total run time was 1.8 min only for each sample, which makes it possible to analyze more than 350 samples per day.