Acid In Human Plasma Research Articles

Determination of free and total valproic acid in human plasma by capillary electrophoresis with contactless conductivity detection

A new approach for the determination of free and total valproic acid in small samples of 140μL human plasma based on capillary electrophoresis with contactless conductivity detection is proposed. A dispersive liquid–liquid microextraction technique was employed in order to remove biological matrices prior to instrumental analysis. The free valproic acid was determined by isolating free valproic acid from protein-bound valproic acid by ultrafiltration under centrifugation of 100μL sample. The filtrate was acidified to turn valproic acid into its protonated neutral form and then extracted. The determination of total valproic acid was carried out by acidifying 40μL untreated plasma to release the protein-bound valproic acid prior to extraction. A solution consisting of 10mM histidine, 10mM 3-(N-morpholino)propanesulfonic acid and 10μM hexadecyltrimethylammonium bromide of pH 6.5 was used as background electrolyte for the electrophoretic separation. The method showed good linearity in the range of 0.4–300μg/mL with a correlation coefficient of 0.9996. The limit of detection was 0.08μg/mL, and the reproducibility of the peak area was excellent (RSD=0.7–3.5%, n=3, for the concentration range from 1 to 150μg/mL). The results for the free and total valproic acid concentration in human plasma were found to be comparable to those obtained with a standard immunoassay. The corresponding correlation coefficients were 0.9847 for free and 0.9521 for total valproic acid.

Silver nanoparticles plasmon resonance-based method for the determination of uric acid in human plasma and urine samples

We have developed a simple and sensitive colorimetric procedure for the quantification of trace amounts of uric acid. It is based on the finding that uric acid in a medium containing ammonia and sodium hydroxide at 65 °C can reduce silver ions to form yellow silver nanoparticles (Ag NPs). These are stabilized in solution by using poly(vinyl alcohol) as a capping agent. The yellow color of the solution that results from the localized surface plasmon resonance of Ag NPs can be observed by the bare eye. The absorbance at 415 nm is proportional to the concentration of uric acid which therefore can be determined quantitatively. The calibration curve is linear in the concentration range from 10 to 200 nM, with a limit of detection of 3.3 nM. The method was successfully applied to the determination of uric acid in human plasma and urine samples.

DETERMINATION OF TOTAL AND UNBOUND CONCENTRATIONS OF VALPROIC ACID IN HUMAN PLASMA BY LIQUID CHROMATOGRAPHY-TANDEM MASS SPECTROMETRY

An analytical method for the determination of valproic acid in human plasma and ultrafiltrated human plasma has been developed and validated. The analytical method lies in the solid phase extraction of valproic acid (VPA), followed by its quantification through an LC-MS method, using benzoic acid as the internal standard. The intra-assay and inter-assay precisions of this technique ranged between 4.29% and 9.91% and its accuracy ranged between 1.00% and 7.73%, for VPA spiked in plasma matrix. For VPA spiked in ultrafiltrated plasma, matrix precisions were from 6.29% to 9.84% and accuracies, from −1.17% to 5.38%. The limit of quantification was set at 1 µg/mL (precision 3.20%, accuracy 11.31%). The linear working range of concentrations was established between 10 and 250 µg/mL for the plasma matrix and between 1 and 100 µg/mL for the ultrafiltrated plasma matrix. The absolute recovery of valproic acid was over 82% in the studied range of concentrations in both matrixes. Valproic acid was found to be stable in both matrixes at the assayed conditions. This method has shown to be suitable for the analysis of free and total valproic acid concentrations in human plasma samples.

Abstract 759: Quantitation of the HDAC inhibitor belinostat (PXD-101) and metabolites in human plasma by a novel LC-MS/MS assay.

Abstract Introduction: Histone deacetylases (HDAC) are frequently deregulated in human cancers and their inhibition allows re-expression of silenced genes. Belinostat is an HDAC inhibitor with in vitro and in vivo activity in multiple malignancies, currently in phase II trials. To date, the pharmacokinetics and metabolism of belinostat have not been adequately characterized. To support an organ dysfunction study and clinical development of belinostat, we developed and validated an LC-MS/MS assay for the sensitive, accurate, and precise quantitation of belinostat and its metabolites belinostat-glucuronide, methylated-belinostat, belinostat amide, 3-ASBA, and belinostat acid in human plasma. Methods: The assay used 50 µL of plasma, [13C6]-belinostat and [D5]-3-ASBA as internal standards and acetonitrile (0.1% TFA) for protein precipitation. A UPLC C18 column was used with a gradient elution from 90:10 to 10:90 water-acetonitrile (0.1% formic acid) over the course of 4 min followed by re-equilibration for 3 min (7 min total run time). Flow rate was 0.5 mL/min. An ABI 4000 tandem MS/MS with ESI positive and negative ionization in MRM mode was used to detect the analytes. Bioanalytical method validation was performed based on FDA guidelines. The applicability of the assay was demonstrated by quantitating belinostat and its metabolites in plasma of a human patient sampled over the course of 24 h. Results: Belinostat eluted at 2.9 min, while all metabolites eluted between 2.7 and 3.6 min. The assay was linear and accurate between 30 and 5000 ng/mL for all analytes; 3 triplicate standard curves assayed on 3 separate days displayed a CV <19% at each of 6 different calibration points (30, 100, 300, 1000, 3000, 5000 ng/mL) for belinostat and CV <29% for the metabolites. The accuracy of the assay was between 101% and 106% at 4 different QC levels (30, 100, 2500, and 4000 ng/mL) for belinostat and between 96% and 117% for metabolites, while the precision was <17% at each concentration for belinostat and <26% for metabolites. Recovery was >69% for belinostat and >64% for metabolites. Belinostat and all metabolites could be quantitated in human plasma after IV administration of belinostat over 30 min. Belinostat glucuronide appeared to be the major metabolite with up to 10-fold exposure relative to belinostat. Conclusion: We have developed a sensitive, accurate and precise LC-MS/MS assay that allows quantitation of belinostat and its 5 metabolites in human plasma. UGT1A1 is responsible for glucuronidation, which appears to be a major metabolic route. This assay will be a valuable tool to assess the pharmacokinetics and metabolism of belinostat in humans, and it is being used to support clinical studies employing belinostat in multiple clinical trials. Supported by Grants U01-CA099168 and N01-CM62209, P30-CA47904 from the NCI, and Spectrum Pharmaceuticals Inc and Topotarget A/S Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 759. doi:1538-7445.AM2012-759

Study of disulfide reduction and alkyl chloroformate derivatization of plasma sulfur amino acids using gas chromatography–mass spectrometry

Four disulfide-reducing agents, dithiothreitol (DTT), 2,3-dimercaptopropanesulfonate (DMPS), and the newly tested 2-mercaptoethanesulfonate (MESNA) and Tris(hydroxypropyl)phosphine (THP), were investigated in detail for release of sulfur amino acids in human plasma. After protein precipitation with trichloroacetic acid (TCA), the plasma supernatant was treated with methyl, ethyl, or propyl chloroformate via the well-proven derivatization-extraction technique and the products were subjected to gas chromatographic-mass spectrometric (GC-MS) analysis. All the tested agents proved to be rapid and effective reducing agents for the assay of plasma thiols. When compared with DTT, the novel reducing agents DMPS, MESNA, and THP provided much cleaner extracts and improved analytical performance. Quantification of homocysteine, cysteine, and methionine was performed using their deuterated analogues, whereas other analytes were quantified by means of 4-chlorophenylalanine. Precise and reliable assay of all examined analytes was achieved, irrespective of the chloroformate reagent used. Average relative standard deviations at each analyte level were ≤6%, quantification limits were 0.1-0.2 μmol L(-1), recoveries were 94-121%, and linearity was over three orders of magnitude (r(2) equal to 0.997-0.998). Validation performed with the THP agent and propyl chloroformate derivatization demonstrated the robustness and reliability of this simple sample-preparation methodology.

A high performance liquid chromatography–tandem mass spectrometric method for the determination of mefenamic acid in human plasma: application to pharmacokinetic study

In the present study, the development and validation of an LC-MS/MS method for quantifying mefenamic acid in human plasma is described. The method involves liquid-liquid extraction using diclofenac as an internal standard (IS). Chromatographic separation was achieved on a Thermo Hypurity C(18) , 50 × 4.6 mm, 5 µm column with a mobile phase consisting of 2 m m ammonium acetate buffer and methanol (pH 4.5 adjusted with glacial acetic acid; 15:85, v/v) at a flow-rate of 0.75 mL/min and the total run time was 1.75 min. Analyte was introduced to the LC-MS/MS using an atmospheric pressure ionization source. Both the drug and IS were detected in negative-ion mode using multiple reaction monitoring m/z 240.0 → 196.3 and m/z 294.0 → 250.2, respectively, with a dwell time of 200 ms for each of the transitions. The standard curve was linear from 20 to 6000 ng/mL. This assay allows quantification of mefenamic acid at a concentration as low as 20 ng/mL in human plasma. The observed mean recovery was 73% for the drug. The applicability of this method for pharmacokinetic studies has been established after successful application during a 12-subject bioavailabity study.

Liquid Chromatography/Tandem Mass Spectrometry for the Simultaneous Determination of Ursodiol and its Major Metabolites, Tauroursodeoxycholic Acid and Glycoursodeoxycholic Acid in Human Plasma

A rapid and sensitive method is described for the quantification of ursodiol and its major metabolites glycoursodeoxycholic acid (GUDCA) and tauroursodeoxycholic acid (TUDCA) in human plasma using single internal standard (Ursodeoxycholic Acid d4). Solid phase extraction was performed and chromatographic separation of 5µL injected sample was achieved using Waters Xterra, 5µm column with a mobile phase comprised of methanol and 5 mM ammonium formate with 0.1 % acetic acid ( 70 : 30, v/v ). The mass spectrometer was used in negative ion mode and multiple reactions monitoring using electro spray ionization mode as an interface. The method was fully validated and the calibration curves were linear over the concentration range of 25.9 to 15300.1 ng/mL for ursodiol, 2.7 to 1587.5ng/mLfor tauroursodeoxycholicacid and 25.4 to 15040.9 ng/mL for glycoursodeoxycholic acid. The method was sensitive and specific, with the lower limit of quantification of 25.9, 2.7 and 25.4 ng/ml for ursodiol, tauroursodeoxycholic acid and glycoursodeoxycholic acid respectively. The present method includes a simple and rapid sample preparation with shorter analysis run time and less flow rate compared to previously reported methods. The method was applied successfully for a bioequivalence study in healthy subjects.

High-performance Liquid Chromatography Method for the Determination of Mycophenolic Acid in Human Plasma and Application to a Pharmacokinetic Study of Mycophenolic Acid Dispersible Tablet

A sensitive and selective high-performance liquid chromatographic-ultraviolet (HPLC-UV) method for the determination of mycophenolic acid (MPA, CAS 24280-93-1) in human plasma has been developed. Sample treatment was based on protein precipitation with a trichloroacetic acid-water (10:90, w/v) solution. The analytical determination was carried out by HPLC with ultraviolet detection at 254 nm. Chromatographic separation was achieved on a C18 column by isocratic elution with acetonitrile-water (pH 4.4) (50:50, v/v) at a flow rate of 1.0 mL/min. The method was linear in the concentration range of 0.2-50.0 microg/mL. The lower limit of quantification (LLOQ) was 0.2 microg/ mL. The intra-day and inter-day relative standard deviations across three validation runs over the entire concentration range were less than 7.05%. The accuracy determined at three concentrations (0.4, 5.0 and 20.0 microg/mL for MPA) was within +/- 10.0%. The method was successfully applied to the evaluation of the pharmacokinetic profile of MPA dispersible tablet in 20 healthy volunteers. The results showed that AUC, C(max) and T1/2 for the test and reference formulations were not significantly different (P > 0.05). The relative bioavailability was 96.42 +/- 15.5%.

Establishment of reliable mass spectra and retention indices library: Identification of fatty acids in human plasma without authentic standards

Gas chromatography mass spectrometry (GC–MS) is routinely employed to analyze small molecules in various samples. The more challenge of GC–MS data processing is to identify the unknown compounds in samples. Mass spectra and retention indices library searching are commonly used method. However, the current libraries are often built through collecting data from different groups. To unknown compounds with similar mass spectra and retention indices (e.g. geometric (cis/trans) isomers), the inaccurate results sometime are supplied. In this case, the costly standard compounds have to be used in every analysis. In this report, taking identification of fatty acids as an example, we proposed a strategy of establishment of special database constructed by equivalent chain length (ECL) values in uniform conditions and mass spectra of fatty acid methyl esters (FAMEs). The mass spectral characteristics were firstly used to identify all expected straight saturated fatty acids, and subsequently calculate the ECL for fatty acids in the sample. Finally, the ECL values of fatty acids in the sample were compared with those of fatty acids in the customized database to identify their structures. The results showed that the method developed in this report could effectively identify similar unknown compounds (FAMEs in the human plasma) after validated by the authentic standards.

A novel RealTime HIV-1 Qualitative assay for the detection of HIV-1 nucleic acids in dried blood spots and plasma

Abbott RealTime HIV-1 Qualitative is an in vitro real-time PCR assay for detecting HIV-1 nucleic acids in human plasma and dried blood spots (DBS). The assay was designed to be used in diagnosis of HIV-1 infections in pediatric and adult patients, with an emphasis on the applicability in resource-limited settings. Use of DBS facilitates specimen collection from remote areas and transportation to testing laboratories. Small sample input requirement facilitates testing of specimens with limited collection volume. The Abbott RealTime HIV-1 Qualitative assay is capable of detecting HIV-1 group M subtypes A–H, group O and group N samples. HIV-1 virus concentrations detected with 95% probability were 80copies/mL of plasma using the plasma protocol, and 2469copies/mL of whole blood using the DBS protocol. The assay detected HIV-1 infection in 13 seroconversion panels an average 10.5 days earlier than an HIV-1 antibody test and 4.9 days earlier than a p24 antigen test. For specimens collected from 6 weeks to 18 months old infants born to HIV-1 positive mothers, assay results using both the DBS and plasma protocols agreed well with the Roche Amplicor HIV-1 DNA Test version 1.5 (95.5% agreement for DBS and 97.8% agreement for plasma).

Method development and validation of an HPLC assay for the detection of hopantenic acid in human plasma and its application to a pharmacokinetic study on volunteers

Summary A reliable and sensitive reversed-phase high performance liquid chromatography (RP-HPLC) with ultraviolet (UV) detection method was developed and validated for the quantification of hopantenic acid in human plasma. Hopantenic acid, with protocatechuic acid as the internal standard (IS), was extracted from plasma samples using a liquid-liquid extraction with methanol. A chromatographic separation was achieved on a Luna C18 column (4.6 mm × 150 mm, 5-μm particle size) and precolumn of the same sorbent (2.0 mm). An isocratic elution, at a flow rate of 1.0 mL min−1, was used with a mobile phase consisting of acetonitrile, water, and 0.03% trifluoroacetic acid. The UV detector was set to 205 nm. The elution times for hopantenic acid and IS were ∼4.3 and 5.4 min, respectively. The calibration curve of hopantenic acid was linear (r > 0.9994) over the range of 0.5–100 μg mL−1 in human plasma. The limit of detection and limit of quantification for hopantenic acid were 0.034 and 0.103 μg mL−1, respectively....

Development and validation of highly sensitive method for determination of misoprostol free acid in human plasma by liquid chromatography–electrospray ionization tandem mass spectrometry: Application to a clinical pharmacokinetic study

A highly sensitive, selective and evaporation free SPE extraction, ESI-LC–MS/MS method has been developed for estimation of misoprostol free acid in human plasma using misoprostol acid-d 5 as an internal standard (IS). The analyte was separated using isocratic mobile phase on reverse phase column and analyzed by MS/MS in the multiple reaction monitoring mode using the respective [M−H] anions, m/ z 367–249 for misoprostol acid and m/ z 372–249 for the IS. The total run time was 5.0 min and the elution of misoprostol acid and misoprostol acid-d 5 (IS) occurred at 3.6 min. The developed method was validated in human plasma with a lower limit of quantification of 2.5 pg/mL. A linear response function was established for the range of concentrations 2.5–1200 pg/mL ( r > 0.998) for misoprostol acid in human plasma. The intra and inter-day precision values for misoprostol acid met the acceptance as per FDA guidelines. Misoprostol acid was stable in the battery of stability studies viz., bench-top, auto-sampler and freeze/thaw cycles. The developed assay method was applied to an oral pharmacokinetic study in humans.

Determination of unmetabolized folic acid in human plasma using affinity HPLC

Background: Folic acid (FA) fortification of food created the need to determine whether fortification elevated concentrations of unmetabolized FA in plasma and whether this form of the vitamin in blood is associated with adverse health outcomes.Objective: The objective of this research was to devise a simple, rapid method for the measurement of unmetabolized plasma FA in epidemiologic studies.Design: We previously used the affinity/HPLC with electrochemical detection method to measure folate distribution in human plasma and red blood cells (RBCs). We modified this method with the inclusion of synthetic ethyltetrahydrofolate as an internal standard and with the use of 2 affinity columns connected in parallel to the analytic column through a switching valve to allow one column to be loaded while the other column was eluted into the analytic column.Results: We identified FA and 5-methyltetrahydrofolate (5-mTHF) by retention time and characteristic response across the channels of the electrochemical detector. Limits of detection were 0.034 pmol for 5-mTHF and 0.027 pmol for FA per injection, and the recovery was 92.2% (5-mTHF) and 98.9% (FA). CVs for samples were 8.1% (within day) and 6.8% (between day) for 5-mTHF and 3.2% (within day) and 5.9% (between day) for FA. Total folate with the use of this method correlated highly (r2 = 0.98, P < 0.001) with values from the microbial assay. The run time for the method was 30 min per sample. Researchers can use this method with longer run times to measure the distribution of folate forms in RBCs.Conclusion: This updated method allows efficient analysis of folate forms in human plasma and tissues without the loss of sensitivity or precision.

Profiling of non-esterified fatty acids in human plasma using liquid chromatography-electron ionization mass spectrometry

This paper focuses on the development of a novel approach to analyze underivatized fatty acids in human plasma. The method is based on liquid-liquid extraction followed by reversed phase liquid chromatography coupled to direct-electron ionization mass spectrometry (LC-Direct-EI-MS). The assay is validated. Calibrations show satisfactory linearity and precision in the investigated range of linearity. Recoveries span from 75% to 104%. The method limits of detection, varying from 0.53 to 5.35 μM, are satisfactory for the quantitation of non-esterified fatty acids (NEFAs) in plasma at physiological levels. The method has been successfully applied to the NEFAs profiling of plasma samples from healthy adult volunteers and subjects affected by diabetes mellitus. Compared with published protocols based on gas chromatography-mass spectrometry and liquid chromatography coupled to electrospray ionization mass spectrometry, this method does not require derivatization and does not show matrix effects, thus simplifying sample preparation procedure and reducing the total time of analysis to approximately 90 min. In addition, Direct-EI-MS allows the acquisition of high-quality NIST library-matchable EI spectra, allowing an easy-to-obtain identification of the target NEFAs.

Simultaneous Determination of the Endogenous Free α-Lipoic Acid and Dihydrolipoic Acid in Human Plasma and Erythrocytes by RP-HPLC with Electrochemical Detection

A highly sensitive, precise, and accurate reversed-phase high-performance liquid-chromatography/electrochemical detection method for simultaneous determination of the endogenous free α-lipoic acid and dihydrolipoic acid in biological matrices was developed and validated. The two analytes were extracted from the samples with acetonitrile/10% metaphosphoric acid solution(aqueous) (50/50 v/v). To determine the total lipoic acid, samples were treated with tris(2-carboxyethyl)phosphine solution in phosphate buffer, pH 2.5 with 85% orthophosphoric acid prior to deproteination. The two analytes were separated on a C18 (150 × 4.6 mm, 5 μm) analytical column using acetonitrile-50 mM phosphate buffer, pH 2.5 with 85% orthophosphoric acid (35/65 v/v) as the isocratic mobile phase pumped at a flow rate of 2.0 mL min−1 at the column oven temperature of 35 °C. The column eluents were monitored at a potential of 0.9 V. These analytes were efficiently resolved in <7 min. The present method was sufficiently robust and specific for simultaneous determination of the two analytes and demonstrated acceptable values for linearity (r 2 = 0.999 in the range of 0.1–500 and 0.25–1,000 ng mL−1 for α-lipoic acid and dihydrolipoic acid, respectively), recovery (>97%), precision (RSD% <2), and sensitivity (on column limit of detection, 150 and 375 fg for α-lipoic acid and dihydrolipoic acid, respectively and limit of quantification: 0.5 and 1.25 pg for α-lipoic acid and dihydrolipoic acid, respectively), indicating that the proposed method was more sensitive, precise, economical, and versatile, and has higher throughput than the previously reported methods for simultaneous determination of the two analytes.

The validation of a simple LC/MS/MS method for determining the level of mevalonic acid in human plasma

A simple plasma extraction method coupled with liquid chromatography-tandem mass spectrometry (LC/MS/MS) detection was developed and validated for the analysis of endogenous mevalonic acid (MVA), a biomarker indicative of the rate of cholesterol biosynthesis, in human plasma samples. The analyte was extracted from the plasma matrix using a straightforward liquid-liquid sample preparation procedure. The extract supernatants were evaporated, reconstituted in aqueous solvent and injected into the LC/MS/MS system without further processing. The chromatographic separation was achieved on a reverse-phase high-performance liquid chromatography column. The accuracy and precision of the method was determined over the concentration range 0.25-25 ng/mL MVA from human plasma extracts in three validation batch runs. Inter-assay precision (%CV) and accuracy (%RE) of the quality control samples were ≤7.00% (at lower limit quality control) and ≤6.10%, respectively. The sensitivity and throughput of this assay was significantly improved relative to previously published methods, resulting in smaller sample requirements and shorter analysis time. Assay results from a clinical study following the oral administration of an exploratory statin demonstrate that this procedure could potentially be used in the investigation of therapies associated with hypercholesterolemia.

Determination of sulphasalazine and its main metabolite sulphapyridine and 5-aminosalicylic acid in human plasma by liquid chromatography/tandem mass spectrometry and its application to a pharmacokinetic study

A simple and sensitive liquid chromatography/positive-ion electrospray ionization mass spectrometry (LC–ESI-MS/MS) method has been developed for the simultaneous determination of sulphasalazine (SASP) and its main metabolite sulphapyridine (SP) and 5-aminosalicylic acid (5-ASA) with 100 μL of human plasma using dimenhydrinate as the internal standard (I.S.). The API-3000 LC–MS/MS was operated under the multiple reaction-monitoring mode (MRM) using the electrospray ionization technique. Protein precipitation process was used to extract SASP, SP, 5-ASA and I.S. from human plasma. The total run time was 9.0 min and the elution of SASP, SP and 5-ASA was at 4.8 min, 2.5 min and 2.0 min, respectively. The separation was achieved with a mobile phase consisting of 0.2% formic acid, 2 mM ammonium acetate in water (mobile phase A) and 0.2% formic acid, 2 mM ammonium acetate in methanol (mobile phase B) by using gradient elution on a XBP Phenyl column (100 mm × 2.1 mm, 5 μm). The developed method was validated in human plasma with a lower limit of quantitation of 10 ng/mL for SASP, SP and 5-ASA, respectively. A linear response function was established for the range of concentrations 10–10,000 ng/mL ( r > 0.99) for SASP and 10–1000 ng/mL ( r > 0.99) for SP and 5-ASA. The intra and inter-day precision values for SASP, SP and 5-ASA met the acceptance as per FDA guidelines. SASP, SP and 5-ASA were stable during stability studies, i.e., long term, auto-sampler and freeze/thaw cycles. The method was successfully applied for the evaluation of pharmacokinetics of SASP, SP and 5-ASA after single oral doses of 250 mg SASP to 10 healthy volunteers.

Determination of Clopidogrel Carboxylic Acid in Human Plasma by LC-MS/MS

Background: Clopidogrel, is a thienopyridine derivative labeled for use to prevent thrombosis after coronary artery stenting. Pharmacokinetics of clopidogrel is studied indirectly by quantification of carboxylic acid which is a major metabolite of Clopidogrel. Objective: The aim of this work is to develop and validate a rapid, simple and sensitive LC/MS/MS assay method for the determination of Clopidogrel carboxylic acid in human plasma using Clopidogrel-D4-carboxylic acid as internal standard. Methods: Analytes was extracted from 200 μl of plasma by a simple liquid-liquid extraction using diethyl ether – n-hexane (80:20, v/v). The chromatographic separations were achieved on a C18 column using Methanol, de-ionized water and formic acid as a mobile phase at flow rate of 0.5 ml/minute. Analysis was monitored by multiple reactions monitoring mode based on m/z transition of 308.10→113 for Clopidogrel carboxylic acid and 312.10→129 for internal standard. Result: The method had a total run time of about 4 minutes. The lower limit of quantification (LLOQ) was 25 ng/ml showing good linearity over the working range of 25 – 3000 ng/ml (r ≥ 0.999). The intra- and inter day accuracies were 90% - 98% and 92.138% - 96.889% respectively (deviation within acceptable range ≤ 10%).Conclusion: It was shown that this method is suitable for pharmacokinetic study following oral administration of Clopidogrel and can be successfully applied to the therapeutic drug monitoring of Clopidogrel in Clopidogrel-treated patients.

A simple, sensitive and efficient assay for the determination of d- and l-lactic acid enantiomers in human plasma by high-performance liquid chromatography

A new method for the simultaneous determination of d- and l-lactic acid in human plasma has been developed using high-performance liquid chromatography (HPLC) with fluorescence detection. This method is based on the reaction of lactic acid with (2 S)-2-amino-3-methyl-1-[4-(7-nitro-benzo-2,1,3-oxadiazol-4-yl)-piperazin-1-yl]-butan-1-one (NBD-PZ-Val) in the presence of O-(7-azobenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU) and N-ethyldiisopropylamine (DIEA) to produce fluorescent diastereomeric derivatives that were easily monitored fluorimetrically at λ ex = 490 nm and λ em = 532 nm. The separation was achieved by use of a C18 analytical column (Synergy Hydro 150 mm × 3 mm i.d., 4 μm). The calibration curve was linear over the on-column concentration range of 10–200 μmol/L for d-lactic acid and 0.5–4.0 mmol/L for l-lactic acid. The sensitivity was good with a limit of detection of 5.24 μmol/L for d-lactic acid and 0.15 mmol/L for l-lactic acid. The analytical method was successfully applied to human plasma samples from normal healthy subjects.

Quantitation of ursolic acid in human plasma by ultra performance liquid chromatography tandem mass spectrometry and its pharmacokinetic study

Ursolic acid is a hydroxy pentacyclic triterpene, which proved to have sedation, anti-inflammatory, antibacterial, antiulcer and anti-cancer activities. An ultra-performance liquid chromatography/tandem mass spectrometry (UPLC/MS/MS) method with high selectivity, sensitivity and throughput has been established and validated for quantitation of total ursolic acid in human plasma. Plasma samples were pretreated by liquid–liquid extraction with ethyl acetate and were chromatographed by an ACQUITY UPLC BEH C8 column (100mm×2.1mm, I.D., 1.7μm) using mobile phase consisting of acetonitrile and 10mM ammonium formate (90:10, v/v) at 0.2mL/min. The duration of chromatography analysis was 3min. The multiple reaction monitoring (MRM) was performed at m/z 455.1→455.0 for ursolic acid and m/z 469.3→425.2 for glycyrrhetinic acid (internal standard, IS) in the negative ion mode with electrospray ionization (ESI) source. The assay showed good linearity over the range of 10–5000ng/mL for ursolic acid in human plasma with a lower limit of quantitation of 10ng/mL. The mean extraction recovery was 73.2±4.5% and the matrix ion suppression ranged from −11.4% to −5.6%. The intra- and inter-day precisions were less than 7.0% and 7.2%, respectively, and the accuracy was within ±2.0%. Ursolic acid was stable during the analysis and the storage period. The validated method has been successfully applied to a pharmacokinetic study after intravenous infusion of Ursolic Acid Nano-liposomes to healthy volunteers.