Inhibitor In Human Plasma Research Articles

High Performance Liquid Chromatography Using UV Detection for the Simultaneous Quantification of the New Non-nucleoside Reverse Transcriptase Inhibitor Etravirine (TMC-125), and 4 Protease Inhibitors in Human Plasma

Etravirine (TMC-125, ETV) is a second-generation non-nucleoside reverse transcriptase inhibitor (NNRTI) that demonstrates potent activity against NNRTI-resistant strains of human immunodeficiency virus type-1 (HIV-1). Thus, ETV has been used in combination with ritonavir-boosted protease inhibitor (PI) and integrase inhibitor for therapy-experienced HIV-1-infected patients. On the other hand, as ETV is a substrate and inducer of cytochrome P450 3A4 (CYP3A4), ETV may induce metabolism of PI and alter the concentrations of co-administered PIs. In order to ensure optimal drug efficacy and prevention of resistance, it is essential to monitor plasma concentrations of ETV and PIs. Here we describe the application of HPLC with UV detection for the simulataneous assay of ETV and 4 PIs, darunavir (DRV), atazanavir (ATV), ritonavir (RTV) and lopinavir (LPV). In this study, the calibration curve of each drug was linear with the average accuracy ranging from 93.6 to 110.9%. Both intra- and interday coefficients of variation for each drug were less than 11.6%. The mean recovery of all drugs ranged from 88.0 to 97.5%. The limit of quantification was 0.04, 0.04, 0.04, 0.05 and 0.07 microg/ml for ETV, DRV, ATV, RTV and LPV, respectively. These results demonstrate that our HPLC-UV method can be used for routine determination of plasma concentrations of ETV and 4 PIs in clinical settings.

Quantitation of Non-Inhibitory Anti-Factor VIII Antibodies in human Plasma

Hemophilia A is a clinically heterogeneous bleeding disorder characterized by the absence of functional factor VIII (FVIII). The most severe complication from exposure to exogenous factor VIII therapy is the development of alloantibodies that neutralize the effect of the therapeutic agent. The standard method for quantitation of FVIII inhibitors in human plasma is by the Nijmegen method, a modification of the Bethesda assay. Our objective was to develop a sensitive and specific fluorescence based immunoassay for the quantitation of anti-FVIII antibodies (FVIIIAb) in human plasma. An affinity purified human anti-FVIIIAb isolated from a hemophilia A patient was used as a calibrator, with a detectability limit of 40±1.5pM. The calibrator and the human plasma anti-FVIIIAb were captured on recombinant FVIII coupled microspheres and probed with mouse anti-human Ig conjugated to R-Phycoerythrin. Citrated human plasma samples from 150 healthy donors and 39 inhibitor-negative hemophilia A subjects were studied and compared to 4 inhibitor-positive hemophilia A plasma samples with inhibitor titers of 1BU (94.6±0.08nM), 11BU (214.3±7.1nM), 106BU (2209.4±84.9nM) and 140BU (2417.7±3.8nM) as measured by the Nijmegen method. Among the 150 normal control plasmas, 4 subjects (3%) were determined as positive (anti-FVIIIAb ≥ 40pM) with concentrations in the range of 0.6nM to 1.9nM, well below that of 1BU (94.6±0.08nM) inhibitor-positive sample. Anti-FVIIIAb was also evaluated in seven commercial inhibitor-free plasma samples reported to contain <1% FVIII activity. Two of the seven plasmas were found to contain anti-FVIIIAb with concentrations of 112.9±1.9nM and 71.1±8.5nM. The concentration of the antibody in both samples was within the range of the measured molar concentration of an inhibitor-positive 1BU titer plasma sample. Thirty two inhibitor-negative hemophilia A subjects were also quantitated for anti-FVIIIAb. Eleven of the 32 samples tested positive for anti-FVIIIAb with molar concentrations in the range of 0.6–20nM. The overall prevalence of the non-inhibitory anti-FVIIIAbs in hemophilia A subjects evaluated by our immunoassay (n=39) is approximately 33% relative to healthy donor population in which 3% were determined as positive (≥40pM) for FVIIIAb.

Quantification of seven nucleoside/nucleotide reverse transcriptase inhibitors in human plasma by high-performance liquid chromatography with tandem mass-spectrometry

A simple analytical method was developed in 100 μL of plasma for the simultaneous assay of the 7 nucleoside/nucleotide reverse transcriptase inhibitors (abacavir, didanosine, emtricitabine, lamivudine, stavudine, tenofovir, and zidovudine) currently used for the treatment of HIV-infected patients. After adding the internal standard, 6-beta-hydroxy-theophyline, plasma samples were precipitated with 500 μL acetonitrile and the supernatants were evaporated to dryness. The residues were reconstituted with 500 μL of water and 10 μL of the extracts were injected in the chromatographic system. The chromatographic separation was performed with a C-18 column and a gradient mobile phase consisting of a mixture of water and acetonitrile, both containing 0.05% formic acid. Analytes quantification was performed by electrospray ionisation triple quadrupole mass-spectrometry in the positive mode using selected reaction monitoring (SRM). Intra- and inter-assay precision and accuracy were lower than 20% for the limit of quantification, and 15% for higher concentrations. The method has been implemented to assess plasma concentrations of patients infected by HIV and was found suitable for therapeutic drug monitoring.

Effect of collision‐activated dissociation gas and collision energy on the fragmentation of dipyridamole and its rapid and sensitive liquid chromatography/electrospray ionization tandem mass spectrometric determination in human plasma

The effect of nitrogen as the collision-activated dissociation (CAD) gas on the fragmentation of dipyridamole was investigated in the range of 10-90 eV collision energy. The results support the collision model reported elsewhere, that the degree of ion fragmentation increases with the increasing mass of the collision gas. A simple, sensitive and high-throughput liquid chromatography/tandem mass spectrometry (LC/MS/MS) method has been developed for the determination of dipyridamole, a platelet aggregation inhibitor in human plasma, using granisetron as internal standard (IS). The method involved liquid-liquid extraction of the analyte and IS from 0.5 mL human plasma with diethyl ether. The chromatographic separation was achieved under isocratic conditions and the ion transitions for dipyridamole (m/z 505.40 --> 429.60) and the IS (m/z 313.10 --> 138.20) were monitored on a triple quadrupole mass spectrometer, operating in positive ion multiple reaction monitoring (MRM) mode. The method was validated over the concentration range 5.1-4499.1 ng/mL for dipyridamole. The method was rugged and rapid with a total run time of 1.2 min. It was successfully applied to a pivotal bioequivalence study in 67 healthy human subjects after oral administration of a 75 mg extended release formulation under fasting condition.

Determination of a novel gamma-secretase inhibitor in human plasma and cerebrospinal fluid using automated 96 well solid phase extraction and liquid chromatography/tandem mass spectrometry

A method for determination of a gamma-secretase inhibitor, cis-3-[4-[(4-chlorophenyl)sulfonyl]-4-(2,5-difluorophenyl)cyclohexyl]propanoic acid ( A), in human plasma and cerebrospinal fluid (CSF) has been developed to support the clinical investigation of compound A for its potential treatment of Alzheimer's disease. The method is based on HPLC with atmospheric pressure chemical ionization tandem mass spectrometric detection (APCI-MS/MS) in the negative ionization mode using a heated nebulizer interface. The addition of phosphoric acid at the ratio of 10–30 μL per milliliter of human plasma or CSF was required during clinical sample collection to stabilize an acylglucuronide metabolite ( C), which was potentially present in human plasma and CSF. Tween 20 (10% solution) was added at the ratio of 20 μL per milliliter of CSF during CSF sample collection to prevent the loss of compound A during the storage of clinical samples. The compound A and its analog internal standard ( B) in treated plasma or CSF were isolated from human plasma or CSF using solid phase extraction (SPE) in the 96 well format. The isolated analyte and internal standard were chromatographed on a Phenomenex Synergi ® Polar RP analytical column (50 mm × 3.0 mm, 4 μm), using a mobile phase consisting of 60/40 (v/v, %) acetonitrile/water at a flow-rate of 0.5 mL/min. Tandem mass spectrometric detection was performed using a Sciex API 3000 tandem mass spectrometer operated in the multiple reaction monitoring (MRM) mode using precursor to product ion transitions of 441 → 175 for A and 469 → 175 for B, respectively. The assays were validated over the concentration range of 0.5–200 ng/mL for human plasma and CSF. Replicate analyses ( n = 5) of spiked standards for both assays yielded a linear response with coefficients of variation less than 7% and accuracy within 5% of the nominal concentrations. In addition, the assays were automated to improve sample throughput by utilizing a Packard Multi PROBEII automated liquid handling system and a Tom-Tec Quadra 96 system. Numerous clinical studies have been supported using these assays.

Alpha-1 Antitrypsin Genotypes in Breast Cancer Patients

Proteolytic enzymes play a significant role in malignancy including, loss of growth regulation, invasiveness and metastases formation. Alpha-1 antitrypsin (A1AT) is a secretory glycoprotein produced mainly in liver and monocytes. It is the most abundant serine protease inhibitor in human plasma. Deficiency of A1AT is an inherited disorder characterized by reduced serum level of A1AT. Protease inhibitor Z (PiZ) and protease inhibitor S (PiS) are the most common deficient genotypes of A1AT. The association of deficient A1AT subtypes with several tumors such as primary liver carcinoma, lung cancer, bladder cancer and malignant hepatoma was reported. This study was aimed to test the relationship between A1AT genotypes Z and S and breast cancer in Jordanian female patients. Blood samples were collected from 111 patients. DNA was isolated and polymerase chain reaction (PCR) was performed to amplify the regions contain the Z and S mutations in exon V, and III, respectively. Genotyping of Z and S alleles was performed by restriction fragment length polymorphism technique using Taq1 restriction enzyme. Our results demonstrated that 100% of the breast cancer patients were homozygous for the normal allele Protease inhibitor MM (PiMM) and no PiZ and PiS genotypes were found. In conclusion, there is no relationship between A1AT deficient genotypes Z and S and breast cancer in Jordanian female patients.

Fast and simultaneous determination of darunavir and eleven other antiretroviral drugs for therapeutic drug monitoring: method development and validation for the determination of all currently approved HIV protease inhibitors and non‐nucleoside reverse transcriptase inhibitors in human plasma by liquid chromatography coupled with electrospray ionization tandem mass spectrometry

For the quantification of all currently approved non-nucleoside reverse transcriptase inhibitors and protease inhibitors, including the new protease inhibitor darunavir and the active nelfinavir metabolite M8, an assay was developed, using liquid chromatography coupled with tandem mass spectrometry. The sample pretreatment consisted of a protein precipitation with a mixture of methanol and acetonitrile using only 100 microL plasma. Chromatographic separation was performed on a reversed-phase C18 column (150 x 2.0 mm, particle size 5 microm) with a quick stepwise gradient using an acetate buffer (pH 5) and methanol, at a flow rate of 0.25 mL/min. The analytical run time was only 10 min. The triple quadrupole mass spectrometer was operated in the positive ion mode and multiple reaction monitoring was used for drug quantification. The method was validated over a range of 0.1 to 20 microg/mL for amprenavir, atazanavir, efavirenz, indinavir, lopinavir, nelfinavir, the active nelfinavir metabolite M8, nevirapine and ritonavir, a range of 0.05 to 10 microg/mL for saquinavir and darunavir and a range of 0.5 to 100 microg/mL for tipranavir, based on observed concentration ranges in patients treated with these drugs. D5-squinavir, D6-indinavir, 13C6-efavirenz and dibenzepine were used as internal standards. The method was proven to be specific, accurate, precise and robust. Accuracies ranged from 88.5% to 102.2% and all precisions were less than 9.5%. Furthermore, the assay demonstrates a high sensitivity for all analytes and the stepwise gradient allows addition of new analytes into the same method. The method is now successfully applied for routine therapeutic drug monitoring and pharmacokinetic studies in HIV-infected patients.

Automated enzyme inhibition assay method for the determination of atorvastatin-derived HMG-CoA reductase inhibitors in human plasma using radioactivity detection

Introduction: A Tecan-based enzyme inhibition assay has been developed for the determination of atorvastatin-derived ‘active’ and ‘total’ (active inhibitors plus atorvastatin lactone and other potential inhibitors following base hydrolysis) 3-hydroxy-3-methylglutaryl-Coenzyme A (HMG-CoA) reductase inhibitor concentrations in human plasma. Atorvastatin is an inhibitor of HMG-CoA reductase, which is a key rate-limiting enzyme in the cholesterol biosynthesis. Previously, atorvastatin-derived HMG-CoA reductase inhibitors were measured via enzyme inhibition assays by manual operation. Methods: In this work, an enzyme assay procedure based on 8-tip Tecan robotics and set-up in a 96-well plate format with customized hardware is presented. Following protein precipitation of the plasma sample, an aliquot of the resulting supernatant is mixed with HMG-CoA reductase and 14C-labeled HMG-CoA prior to incubation. The product, 14C-mevalonic acid, is lactonized, separated from unreacted 14C-substrate, and counted in a liquid scintillation counter. Plasma HMG-CoA reductase inhibitor concentrations are measured against atorvastatin as the standard. Tecan Genesis 150 and 200 robotic workstations were used for the protein precipitation, enzyme incubation, and product separation. Results: The standard calibration range for the assay was 0.4–20 ng eq/mL. Intra-day precision (%CV) data for the calibration standard and quality control (QC) samples ( n = 5 replicates) were both ≤ 8%, with an accuracy between 88 and 113% of nominal values. Initial inter-day precision of the QC samples was ≤ 6%, with an accuracy range of 94–111% of nominal values. Discussion: The assay procedure provides high throughput analysis of clinical samples to support pharmacokinetic studies.

A Simple and Sensitive High-Performance Liquid Chromatographic Method for Quantification of PXD101, a Histone Deacetylase Inhibitor in Human Plasma

PXD101, a new histone deacetylase inhibitor, is currently undergoing phase I/II clinical trials as an anticancer drug. This study describes a simple and sensitive high-performance liquid chromatography ultraviolet method developed for the quantification of PXD101 in human plasma samples to support such trials. Following solid phase extraction at room temperature, the analytes were separated on a 5 mum C18 150 x 2.1 mm column using gradient elution (mobile phase of acetonitrile and 25 mM NaH2PO4, pH 2.8) at a flow rate of 0.5 mL/min and ultraviolet detection at 268 nm. Oxamflatin was used as an internal standard. PXD101 and the internal standard were eluted at about 7.9 min and 13.6 min, respectively. The lower limit of quantification of PXD101 in plasma was 10 ng/mL. The calibration curves for concentrations in the range of 10 to 2,000 ng/mL gave excellent linearity (r = 0.999). The coefficients of variation for intraday and interday assays were all less than 10%. The accuracy of all concentration determinations ranged from 98.0% to 102.0%. There were no problems with PXD101 stability following freeze-thawing, short-term exposure to room temperature, postextraction stability up to 24 hours, and sample storage at -70 degrees C for 3 months. The reported method, with dilution integrity of up to 50 fold, has the requisite sensitivity and is suitable for pharmacokinetic studies of PXD101.

Longitudinal studies of inter-alpha inhibitor proteins in severely septic patients: A potential clinical marker and mediator of severe sepsis*

To determine the clinical relevance and prognostic significance of serial measurement of inter-alpha inhibitor proteins (IalphaIp) in severely septic patients. A laboratory-based study of serial plasma samples over the first 5 days of severe sepsis from a prospective clinical trial. Small business and academic medical center research laboratories. Two hundred sixty-six patients with severe sepsis from a multiple-center phase III clinical trial. None. Inter-alpha inhibitor proteins serve as endogenous serine protease inhibitors in human plasma. The levels of IalphaIp were markedly reduced to a mean value of 290+/-15 microg/mL at the onset of severe sepsis compared with normal plasma levels (617+/-197 microg/mL). Failure of IalphaIp levels to recover over the first 5 days of sepsis was associated with an unfavorable outcome (p<.001). IalphaIp levels were inversely correlated with interleukin-6 levels at study entry and over the first 5 days of management of severe sepsis. IalphaIp levels were significantly lower in women, with increased age, in the presence of multiple organ failure and in patients with intra-abdominal sources of sepsis. Inter-alpha inhibitor proteins are markedly reduced in severe sepsis, and failure of recovery of IalphaIp levels over the course of sepsis is associated with an unfavorable outcome.

Quantitative Determination of Galantamine in Human Plasma by Sensitive Liquid Chromatography--Tandem Mass Spectrometry Using Loratadine as an Internal Standard

A simple, rapid, sensitive, and selective liquid chromatography-tandem mass spectrometry method is developed and validated for the quantitation of galantamine, an acetylcholinesterase inhibitor in human plasma, using a commercially available compound, loratadine, as the internal standard. Following liquid-liquid extraction, the analytes are separated using an isocratic mobile phase on a reverse-phase C18 column and analyzed by mass spectrometry in the multiple reaction monitoring mode using the respective (M+H)+ ions, m/z 288 to 213 for galantamine and m/z 383 and 337 for the internal standard. The assay exhibit a linear dynamic range of 0.5-100 ng/mL for galantamine in human plasma. The lower limit of quantitation is 0.5 ng/mL, with a relative standard deviation of less than 8%. Acceptable precision and accuracy are obtained for concentrations over the standard curve range. A run time of 2.5 min for each sample makes it possible to analyze more than 400 human plasma samples per day. The validated method is successfully used to analyze human plasma samples for application in pharmacokinetic, bioavailability, or bioequivalence studies.

Simultaneous determination of 8 HIV protease inhibitors in human plasma by isocratic high-performance liquid chromatography with combined use of UV and fluorescence detection: Amprenavir, indinavir, atazanavir, ritonavir, lopinavir, saquinavir, nelfinavir and M8-nelfinavir metabolite

A simple, accurate and fast method was developed for determination of the commonly used HIV protease inhibitors (PIs) amprenavir, indinavir, atazanavir, ritonavir, lopinavir, nelfinavir, M8-nelfinavir metabolite and saquinavir in human plasma. Liquid–liquid extraction was used with hexane/ethylacetate from buffered plasma samples with a borate buffer pH 9.0. Isocratic chromatographic separation of all components was performed on an Allsphere hexyl HPLC column with combined UV and fluorescence detection. Calibration curves were constructed in the range of 0.025–10 mg/l. Accuracy and precision of the standards were all below 15% and the lowest limit of quantitation was 0.025 mg/l. Stability of quality control samples at different temperature conditions was found to be below 20% of nominal values. The advantages of this method are: (1) inclusion and determination of the newly approved atazanavir, (2) simultaneous isocratic HPLC separation of all compounds and (3) increased specificity and sensitivity for amprenavir by using fluorescence detection. This method can be used for therapeutic drug monitoring of all PIs currently commercialised and is now part of current clinical practice.

Biological fingerprinting analysis of the interactome of a kinase inhibitor in human plasma by a chemiproteomic approach

In this study, a gel free chemiproteomic method based on chromatography was developed and applied for the biological fingerprinting analysis of complex biological system. p-Aminobenzamidine (ABA), an inhibitor of trypsin-like serine proteases, was immobilized for characterizing their interacting proteins in human plasma. By the proteomic analysis method, 214 proteins were identified with obvious affinity to the immobilized ABA. By searching the sequences of above proteins with consensus patterns of the two active sites, seven proteins belong to trypsin-like serine protease group were found. Based on the Gene Ontology annotation, the identified trypsin-like serine proteases have the function of catalytic activity and calcium ion binding, and are mainly involved in the biological process of blood coagulation. Eight more other proteins related to calcium ion binding and blood coagulation were found. Nearly all of these proteins cannot be identified by directly analyzing the plasma sample demonstrating the chemiproteomics a useful approach to characterize interacting proteins in the low abundance range.

Association of Moderate Alpha-1 Antitrypsin Deficiency with Lung Cancer in the Serbian Population

Alpha-1 antitrypsin (AAT) is an important serine protease inhibitor in human plasma. Its major physiological role is to inhibit neutrophil elastase (NE) in the lower respiratory tract and protect lung tissue from destruction. Recent studies indicated an etiological role of NE in lung cancer development. The aim of this study was to investigate the association of alpha-1 antitrypsin deficiency (AATD) with lung cancer in patients with four different histological types of cancer: squamous cell carcinoma, adenocarcinomas, large cell carcinoma and small cell carcinoma. Phenotyping was carried out by isoelectric focusing (pH 4.2-4.9). We compared the frequency of AATD phenotypes in 186 lung cancer patients with the value obtained in our previous study in a healthy Serbian population (3.7%) using the Fisher exact test. Allele frequencies in patients were Pi *M 0.9677, Pi *Z 0.0215, Pi *S 0.0081 and Pi *other rare 0.0027. Eleven of the 186 lung cancer patients (5.9%) were AATD heterozygotes with moderate deficiencies (PiMZ and PiMS). When this value was compared with AATD heterozygote frequency obtained in the healthy individuals (3.7%), the difference was close to the level of significance (p = 0.055). However, individuals with AATD phenotypes had a higher risk of developing squamous cell lung cancer then those with non-deficient AAT variants (OR = 4.51, 95% CI = 1.66-12.29). Our findings provide evidence of an association between AAT phenotypes with moderate deficiency and squamous cell lung cancer.

First LC–MS/MS electrospray ionization validated method for the quantification of perindopril and its metabolite perindoprilat in human plasma and its application to bioequivalence study

A high throughput bioanalytical method based on solid phase extraction and liquid chromatography-tandem mass spectrometry (LC-MS/MS), has been developed for the estimation of perindopril and its metabolite perindoprilat, an angiotensin-converting enzyme inhibitor in human plasma. Ramipril was used as internal standard (IS). The extraction of perindopril, perindoprilat and ramipril from the plasma involved treatment with phosphoric acid followed by solid phase extraction (SPE) using hydrophilic lipophilic balance HLB cartridge. The SPE eluate without drying were analyzed by LC-MS/MS, equipped with turbo ion spray (TIS) source, operating in the negative ion and selective reaction monitoring (SRM) acquisition mode to quantify perindopril and perindoprilat in human plasma. The total chromatographic run time was 1.5 min with retention time for perindopril, perindoprilat and ramipril at 0.33, 0.35 and 0.30 min. The developed method was validated in human plasma matrix, with a sensitivity of 0.5 ng/ml (CV, 7.67%) for perindopril and 0.3 ng/ml (CV, 4.94%) for perindoprilat. This method was extensively validated for its accuracy, precision, recovery, stability studies and matrix effect especially because the pattern of elution of all the analytes appears as flow injection elution. Sample preparation by this method yielded extremely clean extracts with very good and consistent mean recoveries; 78.29% for perindopril, 76.32% for perindoprilat and 77.72% for IS. The response of the LC-MS/MS method for perindopril and perindoprilat was linear over the range 0.5-350.0 ng/ml for perindopril and 0.3-40 ng/ml for perindoprilat with correlation coefficient, r>/=0.9998 and 0.9996, respectively. The method was successfully applied for bioequivalence studies in human subjects samples with 4 mg immediate release (IR) formulations.

Inter-Alpha-Inhibitor Proteins Are Endogenous Furin Inhibitors and Provide Protection against Experimental Anthrax Intoxication

Inter-alpha-inhibitor protein (IalphaIp) functions as an endogenous serine protease inhibitor in human plasma, and IalphaIp levels diminish rapidly during acute inflammatory states. One potential target for IalphaIp is furin, a cell-associated serine endopeptidase essential for the activation of protective antigen and the formation of anthrax lethal toxin (LT). IalphaIp blocks furin activity in vitro and provides significant protection against cytotoxicity for murine peritoneal macrophages exposed to up to 500 ng/ml LT. A monoclonal antibody (MAb), 69.31, that specifically blocks the enzymatic activity of IalphaIp eliminates its protective effect against LT-induced cytotoxicity. IalphaIp (30 mg/kg of body weight) administered to BALB/c mice 1 hour prior to an intravenous LT challenge resulted in 71% survival after 7 days compared with no survivors among the control animals (P < 0.001). We conclude that human IalphaIp may be an effective preventative or therapeutic agent against anthrax intoxication.

Alpha-1 antitrypsin genotypes in breast cancer patients

Alpha-1 antitrypsin (1-AT) is a secretory glycoprotein mainly produced in the liver and monocytes. It is the most abundant serine protease inhibitor in human plasma. Proteolytic enzymes play a significant role in the expression of the malignant phenotype, including the loss of growth regulation, invasiveness and formation of metastases. Deficiency of 1-AT is an inherited disorder characterized by reduced serum level of 1-AT. Protease inhibitors Z (PiZ) and protease inhibitors S (PiS) are the most common deficient genotypes of 1-AT. The association of deficient 1-AT subtypes with several tumors such as primary liver carcinoma, lung cancer, bladder cancer and malignant hepatoma was reported. This study aimed to determine the incidence of 1-AT genotypes (PiZ and PiS) in breast cancer female patients. Blood samples were collected from 111 patients. DNA was isolated and the PCR technique was performed to amplify the regions contain the Z and S mutations in exon V and exon III, respectively. Genotyping of the Z and S alleles was performed by restriction fragment length polymorphism analysis using the Taq1 restriction enzyme. Our results demonstrated that 100% of the breast cancer patients were homozygous for the normal allele (PiMM) and no PiZ and PiS genotypes were found.

Determination of an investigational HIV integrase inhibitor in human plasma using high performance liquid chromatography with tandem mass spectrometric detection

An HPLC–MS/MS assay for the determination of an HIV integrase inhibitor, 5-(1,1-dioxido-1,2-thiazinan-2-yl)- N-(4-fluorobenzyl)-8-hydroxy-1,6-naphthyridine-7-carboxamide ( I) in human plasma has been developed and validated. Compound I and a stable isotope labeled internal standard ( II) were isolated from 0.5 mL plasma samples by solid phase extraction using an Ansys SPEC C-8 96-well plate. Extracts were separated on a Hypersil BDS C-18 HPLC column (3.0 mm × 50 mm, 3 μm) with a mobile phase consisting of 25 mM ammonium formate pH 3.0:acetonitrile (60:40) vol%/vol% pumped at 0.5 mL/min. A Sciex API 365 mass spectrometer equipped with an atmospheric pressure chemical ionization source was operated in selected reaction monitoring (SRM) mode with the precursor-to-product ion transitions m/ z 431 → 109 ( I) and m/ z 437 → 115 ( II) used for quantitation. The assay was validated over the concentration range of 10–5000 ng/mL and was found to have acceptable accuracy, precision, linearity, and selectivity. The mean extraction recovery from spiked plasma samples was 69%. The intra-day accuracy of the assay was within 4% of nominal and intra-day precision was better than 4% C.V. Following a 200 mg dose of the compound administered to human subjects, concentrations of I ranged from 21.1 to 1500 ng/mL in plasma samples collected up to 12 h after dosing. Inter-day accuracy and precision results for quality control samples run over a 3-month period alongside clinical samples showed mean accuracies of within 6% of nominal and precision better than 3.5% C.V.

Determination of a novel substance P inhibitor in human plasma by high-performance liquid chromatography with atmospheric pressure chemical ionization mass spectrometric detection using single and triple quadrupole detectors

Methods based on high-performance liquid chromatography (HPLC) with atmospheric-pressure chemical ionization (APCI) mass spectrometric (MS) detection using either single (MS) or triple (MS/MS) quadrupole mass spectrometric detection for the determination of (2 R)-[1( R)-(3,5-bis-trifluoromethylphenyl)ethoxy]-3( S)-(4-fluoro-phenyl)morpholin-4-ylmethyl]-5-oxo-4,5-dihydro-[1,2,4]triazol)methyl morpholine (Aprepitant, Fig. 1) in human plasma has been developed. Aprepitant ( I) and internal standard ( II, Fig. 1) were isolated from the plasma matrix buffered to pH 9.8 using a liquid–liquid extraction with methyl- t-butyl ether (MTBE). The analytes were separated on a Keystone Scientific’s Javelin BDS C-8 2 mm×4.6 mm 3 μm guard column coupled to BDS C-8 50 mm×4.6 mm 3 μm analytical column, utilizing a mobile phase of 50% acetonitrile and 50% water containing 0.1% formic acid and 10 mM ammonium acetate delivered at a flow rate of 1 ml/min. The single quadrupole instrument was operated in a single ion monitoring (SIM) mode analyzing the protonated molecules of Aprepitant and II at m/ z 535 and 503, respectively. The triple quadrupole mass spectrometer was operated in multiple reaction monitoring mode (MRM) monitoring the precursor→product ion combinations of m/ z 535→277 and 503→259 for Aprepitant and II, respectively. The linear calibration range for both single and triple quadrupole detectors was from 10 to 5000 ng/ml of plasma with coefficients of variation less than 8% at all concentrations. Both single and triple quadrupole instruments yielded similar precision and accuracy results. Matrix effect experiments performed on both instruments demonstrated the absence of any significant change in ionization of the analytes when comparing neat standards to analytes in the presence of plasma matrix. Both instruments were used successfully to support numerous clinical trials of Aprepitant.

This month in Gastroenterology

This month in Gastroenterology