Validation of an electrospray ionisation LC-MS/MS method for quantitative analysis of telaprevir and its R-diastereomer.

A sensitive high-performance reverse phase liquid chromatography-positive ion electrospray tandem mass spectrometry method was developed and validated for the quantification of telaprevir and its inactive R-diastereomer (VRT-127394) in human plasma. The analytes and the internal standard (telaprevir-d11) were extracted from plasma by liquid-liquid extraction using tert-Butyl methyl ether (TBME). Chromatographic separation was achieved on a reversed-phase Accucore C18 column with a gradient programme consisting of water:ammonia (25%), 100:0.01 (v/v) (mobile phase A) and ACN:MeOH:ammonia (25%), 15:85:0.01 (v/v/v) (mobile phase B). The MS acquisition was performed with selective reaction monitoring mode using the respective [M+H](+) ions, m/z 680.59→322.42 for telaprevir and VRT-127394, and 691.15→110.13 for telaprevir-d11. The assay exhibited a linear dynamic range of 5-5000ng/mL for telaprevir and VRT-127394. Acceptable precision (%RSD<6.5%) and accuracy (94-108%) were obtained for concentrations over the range of the standard curve. A procedure was established to stabilise the plasma to prevent ex vivo interconversion of the isomers.

External validation of the bilirubin-atazanavir nomogram for assessment of atazanavir plasma exposure in HIV-1-infected patients.

Atazanavir increases plasma bilirubin levels in a concentration-dependent manner. Due to less costly and readily available assays, bilirubin has been proposed as a marker of atazanavir exposure. In this work, a previously developed nomogram for detection of suboptimal atazanavir exposure is validated against external patient populations. The bilirubin nomogram was validated against 311 matching bilirubin and atazanavir samples from 166 HIV-1-infected Norwegian, French, and Italian patients on a ritonavir-boosted regimen. In addition, the nomogram was evaluated in 56 Italian patients on an unboosted regimen. The predictive properties of the nomogram were validated against observed atazanavir plasma concentrations. The use of the nomogram to detect non-adherence was also investigated by simulation. The bilirubin nomogram predicted suboptimal exposure in the patient populations on a ritonavir-boosted regimen with a negative predictive value of 97% (95% CI 95-100). The bilirubin nomogram and monitoring of atazanavir concentrations had similar predictive properties for detecting non-adherence based on simulations. Although both methods performed adequately during a period of non-adherence, they had lower predictive power to detect past non-adherence episodes. Using the bilirubin nomogram for detection of suboptimal atazanavir exposure in patients on a ritonavir-boosted regimen is a rapid and cost-effective alternative to routine measurements of the actual atazanavir exposure in plasma. Its application may be useful in clinical settings if atazanavir concentrations are not available.

Comparison of extraction procedures for assessment of matrix effect for selective and reliable determination of atazanavir in human plasma by LC-ESI-MS/MS.

A comparative study with three conventional extraction techniques namely protein precipitation (PP), liquid-liquid extraction (LLE) and solid phase extraction (SPE) has been demonstrated to assess the magnitude of matrix interference by post-column analyte infusion and post extraction analyte spiking for the determination of atazanavir from human plasma. Severe ion suppression observed in PP and to a lesser extent in LLE was circumvented by SPE on LiChrosep Sequence extraction cartridge. Based on these observations a selective, rugged and high throughput SPE-LC-MS/MS method has been developed for reliable determination of atazanavir in human plasma. The chromatographic separation was achieved on a Hypersil Gold C18 (50mm×4.6mm, 5µm) analytical column using 5mM ammonium formate in water:methanol (10:90, v/v) as the mobile phase under isocratic conditions. The method was validated over a wide dynamic concentration range of 10-6000ng/mL. The mean relative recovery and absolute matrix effect across quality controls were 84.9 and 93.2%, respectively. The precision value for relative matrix effect between eight different lots of plasma, expressed as %CV of the slopes of the calibration lines was 2.41. The stability of atazanavir under different storage conditions varied from -8.4 to 5.4%. The method was successfully applied to a bioequivalence study of 300mg atazanavir capsule formulation in 24 healthy Indian males under fasting condition.

Atazanavir metabolism according to CYP3A5 status: an in vitro-in vivo assessment.

The current study was a follow-up to an in vivo study in which atazanavir oral clearance was shown to be dependent on genetically determined CYP3A5 expression status, but only in non-African Americans. The aim of this study was to identify atazanavir metabolites generated by CYP3A5 and to evaluate this metabolite pattern in the African-American versus non-African-American CYP3A5 expressors from the previous study. First, the in vitro metabolism of atazanavir was evaluated using human liver microsomes (HLM) and CYP3A4 and CYP3A5 isoforms. Second, the metabolite pattern generated by CYP3A5 was evaluated in human plasma samples from the previous study. Atazanavir metabolites were analyzed using liquid chromatography-tandem mass spectrometry methods. Metabolite areas under the time-concentration curves (AUCs) were normalized to atazanavir AUC to generate an AUC ratio. Sixteen metabolites were observed in human liver microsomal incubations representing five "phase I" biotransformation pathways. Mono-oxidation products (M1 and M2) were formed by CYP3A5 at a faster rate than CYP3A4 by 32- and 2.6-fold, respectively. This finding was replicated in HLM from a genetically determined CYP3A5 expressor versus nonexpressor. In the in vivo samples, the M1 and M2 AUC ratios were approximately 2-fold higher in CYP3A5 expressors versus nonexpressors (P < 0.05), and the difference was similar in African Americans and non-African Americans. Thus, CYP3A5 produced a unique metabolite "signature" for atazanavir in vitro and in vivo, independent of race. Therefore, other pharmacological factors are likely to explain the apparent lack of effect of genetically determined CYP3A5 expressor status on atazanavir oral clearance in African Americans from the previous study.

Identification of N-acetyl Proline-Glycine-Proline (acPGP) in human serum of adults and newborns by liquid chromatography-tandem mass spectrometry.

BACKGROUND: N-acetyl Proline-Glycine-Proline (acPGP) is a novel neutrophil chemoattractant. However, no studies have been reported to identify the presence of acPGP in human serum. The purpose of our study was to establish a method for measuring acPGP, and to determine whether acPGP is present in human serum. METHODS: Serum samples were obtained from 22 healthy adults and 26 term and preterm newborns. For the sensitive analysis of acPGP, we utilized liquid chromatography-tandem mass spectrometry (LC-MS/MS) with a multiple reaction monitoring (MRM) positive ion mode. RESULTS: The major product ions of acPGP ([M+H](+) ion, m/z 312) appeared at 112 and 140. The MRM (transition: m/z 312/112) chromatogram in human serum showed a single peak with the same retention time as that of authentic acPGP. The calibration curve of authentic acPGP was linear, and our quantitative results indicated high precision. The mean serum acPGP levels in adults and newborns were 6.3 and 18.7 pg/ml, respectively. In newborns, lower birth weight infants had significantly higher serum acPGP levels. CONCLUSIONS: We established a method for the quantification of serum acPGP using LC-MS/MS, and this paper provides the first evidence for the presence of acPGP in human serum of adults and newborns.

First-trimester maternal serum PP13 in the risk assessment for preeclampsia.

OBJECTIVE: The objective of the study was to determine whether first-trimester maternal serum placental protein 13 (PP13) concentrations can be used in the risk assessment for preeclampsia. STUDY DESIGN: This case-control study included 50 patients with preeclampsia and 250 patients with normal pregnancies. Samples were collected between 8 and 13 weeks of gestation. Serum PP13 concentrations were measured by immunoassay and expressed as medians and multiples of the median (MoM) for gestational age. Sensitivity and specificity were derived from receiver-operating characteristic curve analysis. RESULTS: (1) Serum PP13 concentration in the first trimester was significantly lower in patients who developed preterm and early-onset preeclampsia than in those with normal pregnancies; and (2) at 80% specificity, a cutoff of 0.39 MoM had a sensitivity of 100% for early-onset preeclampsia and 85% for preterm preeclampsia. CONCLUSION: Maternal serum first-trimester PP13 appears to be a reasonable marker for risk assessment for preterm preeclampsia but a weak marker for severe preeclampsia at term, and ineffective for identifying mild preeclampsia at term.

Full validation of an analytical method for the HIV-protease inhibitor atazanavir in combination with 8 other antiretroviral agents and its applicability to therapeutic drug monitoring.

Because HIV medications are used in combination, it is important to develop multiplex assays to streamline the therapeutic drug monitoring process and provide rapid turnaround. This article reports full validation of an analytical method that combines atazanavir with 6 HIV-protease inhibitors (indinavir, amprenavir, saquinavir, nelfinavir, ritonavir, and lopinavir) and 2 nonnucleoside reverse transcriptase inhibitors (nevirapine and efavirenz). Using 200 microL of plasma and a simple liquid-liquid extraction method, this analytical method achieved a clean baseline and high extraction efficiencies (90.0% to 99.5%). A Zorbax C-18 (150 x 4.6 mm, 3.5 microm) analytical column was used along with a 27-minute linear gradient elution of the mobile phase to provide sharp peaks at 210 nm. This method was validated over a range of 25 to 10,000 ng/mL and is accurate (90.4% to 110.5%) and precise (precision within a day and between days ranged from 2.3% to 8.3%). Because this method is simple and inexpensive, it may have applicability in countries with low resources.

A novel accurate amplification created restriction site method for determination of the wild type and the precore mutant hepatitis B virus variants.

The most commonly occurring hepatitis B virus (HBV) mutation is the G to A mutation at nucleotide 1896 in the precore region. The aim of this study was to develop a novel accurate amplification created restriction site (ACRS) method for determination of the TGG wild type and the TAG precore mutant HBV variants. Two conserved and consensus specific and diagnostic primers introducing BstXI and XagI cleavage sites were designed in order to determine the G1896 wild type and the A1896 precore mutant HBV variants in all HBV genotypes. The results of the ACRS method were compared with sequencing data. With the ACRS method, three different patterns could be distinguished for the wild type, the precore mutant and mixed infection HBV variants. The results of the ACRS method on 30 HBV isolates revealed the TAG precore mutant in 50% (15/30), the TGG wild type variant in 30% (9/30) and the mixed infection in 20% (6/30). The sequencing data of these samples were in agreement with the ACRS results. The ACRS method is a rapid and cost-effective technique for detecting both the TGG wild type and the TAG HBV precore mutant variants. It can be carried out for follow-up of G1896A precore mutant variant in hepatitis B virus infected subjects at routine molecular diagnostic laboratories.

Serum trypsinogen activation peptide in the assessment of the diagnosis and severity of acute pancreatic damage: a pilot study using a new determination technique.

OBJECTIVES: To evaluate the clinical value of a new direct and competitive immunoassay for trypsinogen activation peptide (TAP) determination in acute pancreatitis (AP). METHODS: The subjects were 34 patients with AP (22 mild, 12 severe), 12 patients with nonpancreatic acute abdominal pain (AA), 11 healthy subjects (HS), and 16 consecutive patients who underwent therapeutic ERCP (ERCP). Serum TAP, amylase, and lipase levels were determined in AP, AA, and HS at their initial observation; the AP patients were also studied for 6 consecutive days after admission. In the ERCP patients, serum TAP, amylase, and lipase levels, as well as urine TAP and amylase levels, were determined before and 6 hours after endoscopy. RESULTS: Serum TAP levels on admission were 0.35 +/- 1.60 OD (mean +/- SD) in AP patients and 0.005 +/- 0.001 OD in AA patients, while HS patients had no detectable serum TAP levels. ERCP patients had no detectable serum TAP levels before and 6 hours after the ERCP, whereas urine TAP concentrations were 1.72 +/- 3.43 OD and 0.75 +/- 1.49 OD before and 6 hours after the execution of the endoscopy, respectively (P = 0.249). The sensitivities and specificities of serum TAP, amylase, and lipase levels in discriminating between AP and AA were 23.5% and 91.7%, 94.1% and 100%, and 97.1% and 100%, respectively, while those used in the assessment of the severity of AP were 29.9% and 73.5%, 38.8% and 81.2%, and 28.4% and 83.6%, respectively. CONCLUSION: TAP is of limited value in assessing the diagnosis and the severity of acute pancreatic damage.

Pharmacological assessment of the antithrombotic activity of the peptide thrombin inhibitor, D-methyl-phenylalanyl-prolyl-arginal (GYKI-14766), in a canine model of coronary artery thrombosis.

The antithrombotic activity of the tripeptide thrombin inhibitor, D-methyl-phenylalanyl-prolyl-arginal (GYKI-14766), was compared to heparin in a model of canine coronary artery thrombosis. Thrombogenesis was initiated by electrolytic injury of the intimal surface of the left circumflex coronary artery. Drug administration was started 15 min before initiation of intimal injury. Clotting times and ex vivo platelet aggregation were determined on citrated blood samples. Gingival template bleeding times were determined. Clotting times (thrombin time; activated partial thromboplastin time, APTT; prothrombin time, PT) increased in a dose-dependent manner with both anticoagulants. The two anticoagulants selectively inhibited thrombin-induced platelet aggregation. GYKI-14766 and heparin were found to delay thrombosis significantly when compared to vehicle-treated animals; minimum effective antithrombotic doses were 0.25 mg/kg/h and 80 U/kg + 30 U/kg/h, respectively. GYKI-14766 (0.25 mg/kg/h) had no effect on template bleeding time, APTT or PT. Heparin (80 U/kg + 30 U/kg/h), however, was associated with a 2.5- to 3.0-min increase in template bleeding time, a 1.8-fold and 1.7-fold increase in APTT and PT, respectively. Antithrombotic efficacy was achieved at doses of GYKI-14766 that did not affect APTT, PT or template bleeding time, whereas antithrombotic efficacy observed with heparin was associated with significant increases in APTT, PT and template bleeding time. These data demonstrate that the tripeptide thrombin inhibitor, GYKI-14766, could potentially prove to be a safer and more effective antithrombotic agent than heparin.