Aliquots Of Plasma Research Articles

Determination of the enantiomeric composition of a new insulin sensitizer in plasma samples from non-clinical and clinical investigations using chiral HPLC with electrospray tandem mass spectrometric detection

Two drug assays were developed and applied to assess the enantiomeric composition of an insulin sensitizer drug in plasma after administration of its racemate to man, and in human and animal plasma and serum samples generated after in vitro experiments. The sample preparation for the assays consisted either of protein precipitation and column-switching, or liquid-liquid extraction and direct injection. Subsequently, both assays employed chiral HPLC coupled to atmospheric pressure ionization mass spectrometry. An interconversion of the racemate to a mixture enriched with the (+)-enantiomer could be confirmed for all species and biological matrices. The individual enantiomers could be quantified in the concentration range 0.5-500 ng/ml, starting with a 100-microl plasma aliquot. Inter- and intra-assay precision and accuracy were in the range 0.1-7.9 and 88.8-106.0%, respectively. Run times of 5 min for a single sample allows the analysis of more than 200 samples overnight.

Development of a liquid chromatographic–tandem mass spectrometric method with precolumn derivatization for the determination of dencichine in rat plasma

Dencichine is a nonprotein amino acid used for hemostasis. A very sensitive method was developed for the determination of dencichine concentration in rat plasma using a derivatization step to enhance signal intensity. The method consisted of a protein precipitation extraction followed by derivatization with 10 M hydrochloric acid–methanol (10:90, v/v) and analysis by liquid chromatography coupled with tandem mass spectrometry (LC–MS–MS) via atmospheric pressure chemical ionization (APCI) source. The separation was achieved using a XDB C 8 analytical column with an isocratic mobile phase composed of methanol–water–formic acid in the ratio of 35:65:0.5 (v/v/v). Signal intensity of the dencichine derivative was increased up to 50-fold as compared to the underivatized dencichine in positive APCI mode. The matrix effect was also investigated. The method has a lower limit of quantification of 20 ng/ml for a 100 μl plasma aliquot. The intra- and inter-day precision (R.S.D.), calculated from quality control (QC) samples, was less than 6.3%. The accuracy (R.E.), determined using QC samples, ranged from −0.2 to 3.9%. The method offered increased sensitivity, selectivity and speed of analysis over existing method. The method was utilized to support clinical pharmacokinetic studies of dencichine in rat following intravenous administration. It indicated that this LC–MS–MS method is useful for pharmacokinetic studies of dencichine in animals, because it enabled the serial determination of plasma concentration of dencichine in rats.

Analysis of ADAMTS13 Activity in Plasma Products Using a Modified FRETS-VWF73 Assay.

INTRODUCTION: Many cases of idiopathic thrombotic thrombocytopenic purpura (TTP) are characterized by very low levels of the metalloprotease ADAMTS13, presumably resulting in dysregulation of platelet/vWF interactions and microvascular thrombosis. Treatment for TTP begins with therapeutic plasma exchange (TPE), allowing replacement of ADAMTS13 and removal of autoantibody, when present. Fresh frozen plasma (FFP) and plasma cryoprecipitate reduced (also known as cryoprecipitate poor plasma [CPP]) have been used as replacement fluids. While current AABB standards allow thawed FFP and CPP components to be stored at 1–6°C for up to 5 days before use, the amount of ADAMTS13 activity in these products has not previously been measured nor has its stability been evaluated over the 5 days. Given the labor-intensive nature of our standard collagen-binding assay for ADAMTS13 activity, we chose to analyze ADAMTS13 activity in FFP and CPP components using a modified FRETS-VWF73 assay recently developed in our laboratory. Preliminary data indicate that the two assays correlate well.METHODS: Ten whole blood (WB) units (5 group O and 5 group A) were centrifuged within 8 hours of collection. Prior to freezing of the FFP, an aliquot of plasma was obtained from each component. The aliquots and FFP components were frozen at <-18°C. Following AABB guidelines, each FFP unit was processed into cryoprecipitated AHF and CPP components. The FFP aliquots and their related CPP components were stored frozen at <-18°C for 46 days and then thawed per AABB standards. Once thawed, aliquots and components were placed in a 1–6°C monitored refrigerator for 30 minutes. Each FFP aliquot (approximately 6 ml) was divided in half; one tube (day 0) was frozen at <-70°C, while the other was stored at 1–6°C until expiration of the product (day 5) and then frozen at <-70°C. Samples were sterilely collected from each CPP unit (mean volume 235 ml) 30 minutes after thaw (day 0), and then every 24 hours until expiration (days 1–5). Immediately after sampling, all CPP samples were stored at <-70°C until assayed. Day 0 and Day 5 FFP and CPP samples derived from the same WB units were analyzed for ADAMTS13 activity using a modified VWF73 FRET-labeled peptide assay (Kokame, 2005).RESULTS: Using the 2-sample t-test assuming equal variances, no statistical difference in ADAMTS13 activity was observed between the FFP and CPP products, nor was there a statistical difference observed between day 0 and day 5 samples (Table). Mean levels of ADAMTS13 activity were statistically similar to 20 normal donor (Precision BioLogic) samples (127 ±27 v. 125 ±24, p=NS). However, the mean activity level in group O units was statistically higher when compared to group A units (149 ±13 v. 104 ±16, p <0.00001).ADAMTS13 Activity*Plasma Type (n)Day 0Day 5pTotal FFP (10)127 ± 24122 ± 28NSFFP A (5)106 ± 12108 ± 23NSFFP O (5)148 ± 14144 ± 22NSTotal CPP (5)126 ± 29123 ± 25NSCPP A (5)102 ± 19102 ± 16NSCPP O (5)151 ± 12145 ± 11NS*Mean ± SDCONCLUSION: Our data indicate that both FFP and CPP products have similar ADAMTS13 activity, suggesting that both products should be equally effective in restoring ADAMTS13 activity in TTP patients. The data also indicate that ADAMTS13 is stable at 1–6°C, showing consistent efficacy of thawed FFP and CPP products for ADAMTS13 replacement throughout the refrigerated shelf life of the thawed product. These results reinforce previously reported data that there is a difference in ADAMTS13 activity between blood group O and A individuals.

Extra-Cellular DNA in Cord Blood (CB) Plasma: Use for Sample Identification [short Tandem Repeat (STR) Analysis] and for Infectious Disease Screening in CB Banks.

Introduction: A commitment of CB banks is to test CB units and maternal blood for multiple genetic and infectious disease markers. As a consequence, several aliquots of blood, mononuclear cells and plasma are taken during processing for immediate and delayed testing. Whereas control of identity between aliquots containing cells can be performed by DNA assays, identity controls for plasma aliquots rely only on bar-coded label systems. We hypothesize that there is soluble extra-cellular DNA (EC-DNA) and that it can be used for identity control of plasma aliquots. The aims of our study were;to determine the concentration of EC-DNA in CB-plasma,to evaluate whether genetic identity testing by STR analysis is feasible andto investigate whether EC-DNA allows infectious disease screening by nucleic acid testing (NAT).Methods: CB (n=20) was collected, processed (Rubinstein et al, PNAS 1995) and EC-DNA from 200 μl of 0.45μm-filtered plasma was extracted both with the Qiagen and the High Pure Viral Nucleic Acid method (Roche). Presence of EC-DNA was evaluated by detection of HLA-DR and ALU gene sequences and by STR analysis (AmpFlSTR-Profiler Plus-Applied Biosystems). Purified HBV was added to CB and peripheral blood (PB) plasma, EC-DNA recovered as above and HBV-DNA assessed by real time PCR.Results: All 20 samples were positive for HLA-DR by PCR and for ALU sequence by real time PCR, confirming the presence of EC-DNA in CB-plasma in concentrations of 0.022 +/− 0.012 ng/μl at the time of CB collection, rising to 0.124 +/− 0.025 ng/μl 33 hrs later; p<0.016. Concentrations observed in CB-plasma were higher than those of adult donor plasma (PB-plasma 0.005+/−0.0065 ng/μl). STR analysis was validated on cellular DNA and, when applied for EC-DNA, showed good signal strengths and low backgrounds, allowing accurate automatic allele identification with no manual corrections (genemapper software, Applied Biosystems). An increase in STR-PCR product concentration was observed when we analyzed EC-DNA samples from CB after 33hs (7017.6 +/− 2014 RFU/μl at time of CB collection and 30290.9 +/− 3164 RFU/μl after 33 hs; RFU = relative fluorescence units). HBV was recovered from all spiked units, in correspondence with the viral concentrations added. Recovery of HBV was 96 +/− 21% with high and low viral concentrations. Three mothers with HBAg/HBcore positive had also HBV-DNA in PB-plasma. Six with anti-HBcore only and 70 with anti-HBs were negative. HBV-DNA was also negative in CB-plasma for all seventy nine respective newborns.Conclusions: there is infant EC-DNA in CB-plasma and viral nucleic acids can be obtained using the same extraction method. There is an increase of EC-DNA in CB over time that may reflect cell death. STR assay of EC-DNA can be a useful molecular tool for the identification of plasma aliquots used for infectious disease testing in CB banks.

HDLs in apoA-I transgenic Abca1 knockout mice are remodeled normally in plasma but are hypercatabolized by the kidney

Patients homozygous for Tangier disease have a near absence of plasma HDL as a result of mutations in ABCA1 and hypercatabolize normal HDL particles. To determine the relationship between ABCA1 expression and HDL catabolism, we investigated intravascular remodeling, plasma clearance, and organ-specific uptake of HDL in mice expressing the human apolipoprotein A-I (apoA-I) transgene in the Abca1 knockout background. Small HDL particles (7.5 nm), radiolabeled with (125)I-tyramine cellobiose, were injected into recipient mice to quantify plasma turnover and the organ uptake of tracer. Small HDL tracer was remodeled to 8.2 nm diameter particles within 5 min in human apolipoprotein A-I transgenic (hA-I(Tg)) mice (control) and knockout mice. Decay of tracer from plasma was 1.6-fold more rapid in knockout mice (P < 0.05) and kidney uptake was twice that of controls, with no difference in liver uptake. We also observed 2-fold greater hepatic expression of ABCA1 protein in hA-I(Tg) mice compared with nontransgenic mice, suggesting that overexpression of human apoA-I stabilized hepatic ABCA1 protein in vivo. We conclude that ABCA1 is not required for in vivo remodeling of small HDLs to larger HDL subfractions and that the hypercatabolism of normal HDL particles in knockout mice is attributable to a selective catabolism of HDL apoA-I by the kidney.

Synthetic LXR agonists increase LDL in CETP species

Liver X receptor (LXR) nuclear receptors regulate the expression of genes involved in whole body cholesterol trafficking, including absorption, excretion, catabolism, and cellular efflux, and possess both anti-inflammatory and antidiabetic actions. Accordingly, LXR is considered an appealing drug target for multiple indications. Synthetic LXR agonists demonstrated inhibition of atherosclerosis progression in murine genetic models; however, these and other studies indicated that their major undesired side effect is an increase of plasma and hepatic triglycerides. A significant impediment to extrapolating results with LXR agonists from mouse to humans is the absence in mice of cholesteryl ester transfer protein, a known LXR target gene, and the upregulation in mice but not humans of cholesterol 7alpha-hydroxylase. To better predict the human response to LXR agonism, two synthetic LXR agonists were examined in hamsters and cynomolgus monkeys. In contrast to previously published results in mice, neither LXR agonist increased HDL-cholesterol in hamsters, and similar results were obtained in cynomolgus monkeys. Importantly, in both species, LXR agonists increased LDL-cholesterol, an unfavorable effect not apparent from earlier murine studies. These results reveal additional problems associated with current synthetic LXR agonists and emphasize the importance of profiling compounds in preclinical species with a more human-like LXR response and lipoprotein metabolism.

27: Development of a Multiinstitutional Chronic Pelvic Pain Registry

There is a severe lack of multi-institutional collaboration and general research into etiologies for and optimal treatments of chronic pelvic pain. Our objective was to develop a registry of chronic pelvic pain patients to be used by multiinstitutional researchers. The registry is a custom built web-based application that utilizes Adobe Coldfusion version 5 middleware connecting to an Oracle 9i database. Chronic pelvic pain centers and institutions in multiple countries. Chronic pelvic pain patients as defined by ACOG. Data are entered by research staff anywhere they have internet access. Research staff and physicians collect biological specimens which are stored and logged by staff. The registry currently contains 760 serum, plasma, and buffy coat aliquots from 46 subjects. Additional collection of biological specimens will include diseased and normal peritoneum, peritoneal fluid, endometrium, and myometrium. The registry is a HIPAA compliant, secure environment, accessible from any internet connection. User access is determined by the NYU research team. The online version of the registry has been completed and is currently in use at the NYU Medical Center. It is a highly scalable and adaptable online database. Specific protocols can be carried out within this database. The research possibilities within this environment are limitless.

Plasma appearance of labeled β-carotene, lutein, and retinol in humans after consumption of isotopically labeled kale

The bioavailability of carotenoids from kale was investigated by labeling nutrients in kale with 13C, feeding the kale to seven adult volunteers, and analyzing serial plasma samples for labeled lutein, beta-carotene, and retinol. Ingested doses of labeled carotenoids were 34 micromol for beta-carotene and 33 micromol for lutein. Peak plasma concentrations, areas under the plasma concentration-time curves (AUCs), and percentages of dose recovered at peak plasma concentrations were calculated. Average peak plasma concentrations were 0.38, 0.068, and 0.079 microM for [13C]lutein, [13C]beta-carotene, and [13C]retinol, respectively. Average AUC values (over 28 days) were 42.8, 13.6, 13.2 microM h for [13C]lutein, [13C]beta-carotene, and [13C]retinol, respectively. Percentages of dose recovered at peak plasma concentrations were 3.6, 0.7, and 0.7% for [13C]lutein, [13C]beta-carotene, and [13C]retinol, respectively. A positive relationship was observed between baseline plasma retinol levels and [13C]retinol plasma response. It is possible that this relationship was mediated either through some aspect of beta-carotene absorption or via the common pathways of metabolism for postdose and endogenous retinoid.

Ferrocene-based Diels–Alder derivatization for the determination of 1α-hydroxyvitamin D 3 in rat plasma by high-performance liquid chromatography–electrospray tandem mass spectrometry

A specific and sensitive liquid chromatography–electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) method has been developed for the determination of 1α-hydroxyvitamin D 3 (1αOHD 3) in rat plasma. A new ferrocene-based Cookson-type reagent, 4-ferrocenylmethyl-1,2,4-triazoline-3,5-dione (FMTAD), designed and synthesized to be highly sensitive to vitamin D analogs in ESI, considerably improved the detection limit with 250 fg (359 amol)/injection. 1αOHD 3 in rat plasma was extracted with acetonitrile and then purified using Oasis HLB 96-well plates. After the precolumn derivatization with FMTAD, samples were subjected to LC/ESI-MS/MS employing a column-switching system. This method achieved a lower limit of quantitation of 5 pg from 0.1-mL plasma aliquots and 200-fold sensitivity of that without derivatization. The calibration curve (0.05–15 ng/mL) exhibited acceptable linearity ( r > 0.9966), intraassay precision ranged from 3.8 to 9.6%, interassay precision ranged from 3.0 to 17.0%, and accuracy was within 81.4–112.0%. This FMTAD derivatization method is considered very useful for determination of vitamin D analogs in ESI and applicable for biological samples.

Development of a liquid chromatography/tandem mass spectrometry assay for the quantification of rabeprazole in human plasma

A simple and sensitive liquid chromatography/tandem mass spectrometry method, employing electrospray ionization, has been developed and validated to quantify rabeprazole in human plasma using omeprazole as the internal standard. The method was validated to demonstrate the specificity, lower limit of quantification, accuracy, and precision of measurements. Selected reaction monitoring was specific for rabeprazole and omeprazole (the internal standard, IS); no endogenous materials interfered with the analysis of rabeprazole and IS from blank plasma. The assay was linear over the concentration range 0.2-200 ng/mL using a 2 microL aliquot of plasma. The correlation coefficients for the calibration curves ranged from 0.9988-0.9994. The intra- and inter-day precision, calculated from quality control samples, were less than 6.65%. A mixture of methanol and water (50:50) was used as the isocratic mobile phase, with 0.1% of formic acid in water, that did not affect the stability of rabeprazole or IS. A simple sample preparation method of protein precipitation with methanol was chosen. The method was employed in a pharmacokinetic study after oral administration of 20 mg rabeprazole to 24 healthy volunteers.

Heterozygous mutation of ataxia-telangiectasia mutated gene aggravates hypercholesterolemia in apoE-deficient mice

Individuals with a heterozygous mutation at the ataxia-telangiectasia mutated gene (ATM) have been reported to be predisposed to ischemic heart disease. This report examined for the first time the effect of a heterozygous ATM mutation (ATM(+)(/-)) on plasma lipid levels and atherosclerosis intensity using ATM(+/-), ATM(+)(/+) (wild type), ATM(+)(/+)/LDLR(-)(/-) (low density lipoprotein receptor knockout), ATM(+)(/-)/LDLR(-)(/-), ATM(+)(/+)/ApoE(-)(/-) (apolipoprotein E knockout), and ATM(+)(/-)/ApoE(-)(/-) mice. Our data demonstrated that the plasma cholesterol and triglyceride levels in ATM(+)(/-) and ATM(+)(/-)/LDLR(-)(/-) mice were approximately the same as those in ATM(+)(/+) and ATM(+)(/+)/LDLR(-)(/-) control mice, respectively. In contrast, the plasma cholesterol level was significantly higher in ATM(+)(/-)/ApoE(-)(/-) mice than in ATM(+)(/+)/ApoE(-)(/-) control mice. In addition, the ATM(+)(/-)/ApoE(-)(/-) mice showed higher plasma apoB-48 levels, slower clearance for plasma apoB-48-carrying lipoproteins, and more advanced atherosclerotic lesions in the aorta compared with the ATM(+)(/+)/ApoE(-)(/-) mice. These novel results suggest that the product of ATM is involved in an apoE-independent pathway for catabolism of apoB-48-carrying remnants; therefore, superimposition of a heterozygous ATM mutation onto an ApoE deficiency background reduces the clearance of apoB-48-carrying lipoproteins from the blood circulation and promotes the formation of atherosclerosis.

Codon 12 K-ras mutations in plasma DNA are not an indicator of disease in non-small-cell lung cancer (NSCLC) patients

7204 Background: Overexpression or mutational activation of the K-ras gene, mostly in codon 12, has been found in 30–80% of NSCLCs. Cell-free circulating DNA genetically identical to that of the primary tumor has been detected in the plasma of cancer patients. K-ras mutations were examined in plasma DNA and paired tumor tissue specimens of patients with NSCLC and also in plasmas from a different cohort of NSCLC patients and healthy control subjects. Methods: An overall number of 48 NSCLC patients and 40 controls participated in the study, after written consent. Peripheral blood was collected in EDTA-coated Vacutainers. The DNA was purified from plasma aliquots of 12 NSCLC and the corresponding tumor tissue samples. The plasma DNA was isolated using both the Qiagen method and the modified Guanidine/Promega Resin (G/R) method according to Wang et al. (Kopreski et al. J Natl Cancer Inst 2000; 92:918–23; Wang et al. Clin Chem 2004; 50:211–3). The DNA was also isolated by the Qiagen method from an additional 76 plasma samples (36 NSCLC and 40 controls, respectively). The isolated DNA was amplified by means of a PCR method that enriches for mutations in codon 12 of the K-ras gene. The assay employs simultaneous restriction enzyme digestion and PCR amplification and is similar to previously reported assays described by Kopreski et al. All amplification assays included a K-Ras mutation-positive control. Results: The presence of K-ras mutations in cancer tissue showed no correlation with the corresponding plasma sample, either extracted by the Qiagen or by the G/R Method: Codon 12 K-ras mutations were present in 2 out of 12 tissue samples, while no mutation or two mutations different from those identified in the corresponding tumor tissue were detected in the plasma using the Quiagen and G/R methods, respectively. K-ras mutations were detected in 15 plasma samples collected from the additional 36 NSCLC patients (41.7%) as well as from 12/40 controls (30.0%). Conclusions: Our results do not support the use of codon 12 K-ras mutations in plasma DNA as a diagnostic marker in NSCLC. (This study was funded by the Assessorato Sanità Regione Piemonte - Progetti Ricerca Sanitaria Finalizzata 2003) No significant financial relationships to disclose.

Simultaneous Determination of Pantoprazole and Its Two Metabolites in Dog Plasma by HPLC

A simple and sensitive high-performance liquid chromatography (HPLC) method is developed and validated for simultaneous determination of pantoprazole and its two metabolites (pantoprazole sulfone and pantoprazole thioether) in dog plasma and applied to a pharmacokinetic study in Beagle dogs. Following a protein precipitation procedure, the samples are separated using reversed-phase HPLC (C18) by a gradient of acetonitrile and ammonium acetate (pH 6.0) at a flow rate of 1.0 mL/min and quantitated using UV detection at 290 nm. Omeprazole is selected as the internal standard. The method has a lower limit of quantitation of 0.025 microg/mL for pantoprazole and its two metabolites, using 0.1-mL aliquots of plasma. The linear calibration curves are obtained in the concentration range of 0.025-10.0 microg/mL for three analytes. The intra- and interrun precision (relative standard deviation), calculated from quality control (QC) samples, is less than 13% for three analytes. The accuracy determined from QC samples is between -6.4% and 12%.

Qualitative Determination of Synthetic Analogues of Insulin in Human Plasma by Immunoaffinity Purification and Liquid Chromatography−Tandem Mass Spectrometry for Doping Control Purposes

Synthetic insulins such as Humalog Lispro, Novolog Aspart, or Lantus Glargine, are commonly employed for the treatment of insulin-dependent diabetes mellitus owing to convenient handling and fast or prolonged bioavailability. However, the misuse of insulin in sports has been reported often, and the international doping control system requires a reliable and robust assay to determine the presence or absence of related drugs prohibited by the World Anti-Doping Agency. Qualitative evidence of administered substances, which is preferably obtained by mass spectrometry, is of utmost importance. Plasma specimens of 2 mL were fortified with three synthetic insulin analogues and purified by immunoaffinity chromatography, and extracts were analyzed by microbore liquid chromatography and tandem mass spectrometry. Product ion scan experiments of intact proteins enabled the differentiation between endogenously produced insulin and its synthetic analogues by collisionally activated dissociation of multiply charged precursor ions. This top-down sequencing-based assay allows the assignment of individual fragment ions, in particular, of those comprising modifications that are originating from C-termini of B-chains. Recoveries of synthetic insulins from plasma aliquots ranged from 91 to 98%, and detection limits were accomplished at 0.5 ng/mL for all target analytes.

Determination of talinolol in human plasma using automated on-line solid phase extraction combined with atmospheric pressure chemical ionization tandem mass spectrometry

A specific LC–MS/MS assay was developed for the automated determination of talinolol in human plasma, using on-line solid phase extraction system (prospekt 2) combined with atmospheric pressure chemical ionization (APCI) tandem mass spectrometry. The method involved simple precipitation of plasma proteins with perchloric acid (contained propranolol) as the internal standard (IS) and injection of the supernatant onto a C 8 End Capped (10 mm × 2 mm) cartridge without any evaporation step. Using the back-flush mode, the analytes were transferred onto an analytical column (XTerra C 18, 50 mm × 4.6 mm) for chromatographic separation and mass spectrometry detection. One of the particularities of the assay is that the SPE cartridge is used as a column switching device and not as an SPE cartridge. Therefore, the same SPE cartridge could be used more than 28 times, significantly reducing the analysis cost. APCI ionization was selected to overcome any potential matrix suppression effects because the analyte and IS co-eluted. The mean precision and accuracy in the concentration range 2.5–200 ng/mL was found to be 103% and 7.4%, respectively. The data was assessed from QC samples during the validation phase of the assay. The lower limit of quantification was 2.5 ng/mL, using a 250 μL plasma aliquot. The LC–MS/MS method provided the requisite selectivity, sensitivity, robustness accuracy and precision to assess pharmacokinetics of the compound in several hundred human plasma samples.

Determination of 2‐methoxyestradiol in human plasma, using liquid chromatography/tandem mass spectrometry

A liquid chromatography/tandem mass spectrometric (LC/MS/MS) assay was developed for the quantitative determination of 2-methoxyestradiol (2ME2) in human plasma. Sample pretreatment involved liquid-liquid extraction with ethyl acetate of 0.3-mL aliquots of plasma spiked with the internal standard, deuterated 2ME2 (2ME2-d5). Separation was achieved on a Zorbax Eclipse C18 column (2.1 x 50 mm, i.d., 5 microm) at room temperature using a gradient elution with methanol and water at a flow rate of 0.25 mL/min. Detection was performed using atmospheric pressure chemical ionization MS/MS by monitoring the ion transitions from m/z 303.1 --> 136.8 (2ME2) and m/z 308.1 --> 138.8 (2ME2-d5). Calibration curves were linear in the concentration range of 1-100 ng/mL. The accuracy and precision values, obtained from three different sets of quality control samples analyzed in quintuplicate on four separate occasions, ranged from 105-108% and from 3.62-5.68%, respectively. This assay was subsequently used for the determination of 2ME2 concentration in plasma of a patient with cancer after a single oral administration of 2ME2 at a dose of 2200 mg.

Determination of potato carboxypeptidase inhibitor in African Green Monkey plasma using 96-well SPE and LC–MS/MS

Potato carboxypeptidase inhibitor (CPI), a peptide with multiple isoforms (MW > 4000 Da) was determined from African Green Monkey plasma using a PE Sciex API-3000 LC–MS/MS in the positive ionization mode with the turbo ionspray interface (450 °C). Samples were prepared using an Oasis MCX 96-well solid phase extraction plate and chromatographed on an Allure C 18 HPLC Column (50 mm × 1.0 mm, 5 μm) using gradient elution. Upon analysis of the extracts using LC–MS/MS, the concentration of CPI was calculated using a single MS/MS transition ( m/z 830.5 → 221.0) that was reflective of the mass concentration (μg/mL) of main the CPI isoforms present in plasma from monkeys after they were given an intravenous dose of CPI. The assay was linear for CPI over concentrations of 0.05–10 μg/mL when extracting 200-μL aliquots of African Green Monkey plasma. The assay was applied to the determination of CPI in African Green Monkey plasma samples in two separate analytical runs (correlation of standard curves, r 1 = 0.9991 and r 2 = 0.9953). Quality control (QC) samples were run at 0.05, 0.1, 0.2, 0.5, 1.0, 2.0 and 5.0 μg/mL for each assay. Average ranges ( n = 12) for accuracy and precision for all concentrations of QCs during the two runs were 92.0–102.0% of expected potency and 10.4–21.8% (coefficient of variations), respectively.

Development of a high-performance liquid chromatography–tandem mass spectrometry method for the determination of flurogestone acetate in ovine plasma

Flurogestone (FGA) is a synthetic progesterone, with a progestational action higher than that of progesterone itself. It is intended for vaginal use in large animals to induce oestrus synchronization. A quantitative method for the analysis of flurogestone acetate (FGA) in ovine plasma by high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) has been developed. After the incorporation of megestrol acetate (MGA) as internal standard (IS) and followed by a liquid–liquid extraction from plasma, FGA and MGA were chromatographed using a reverse-phase HPLC column and detected by tandem mass spectrometry with a TurboIonSpray ® source. Multiple reaction-monitoring (MRM) mode was used for the quantitative determination of FGA in ovine plasma. The precursor ions [ M + H] + at m/ z 407.2 and 385.1 for FGA and MGA, respectively, produced product ions at m/ z 267.1/285.1 for FGA and m/ z 267.1/224.0 for MGA. The validated concentration range was 0.2–5.0 ng/ml based on 500 μl plasma aliquots. The lower limit of quantitation was 0.2 ng/ml. Fully validated selectivity, accuracy, precision and reproducibility criteria for routine use in pharmacokinetic studies were demonstrated.

Viral kinetics in hepatitis C or hepatitis C/human immunodeficiency virus-infected patients

Kinetic modeling of hepatitis C virus (HCV) response to interferon (IFN)-based therapy provides insights into factors associated with treatment outcomes. HCV/human immunodeficiency virus (HIV)-co-infected patients show lower response rates vs. HCV-monoinfected patients. Reasons for this remain unclear. This study evaluated kinetic parameters and treatment responses in co-infected vs monoinfected patients. Co-infected patients were randomized within a US multicenter trial (ACTG 5071) to receive pegylated-interferon (PEG-IFN) alfa-2a + ribavirin vs. IFN alfa-2a + ribavirin. Monoinfected controls were matched prospectively for treatment, genotype, age, sex, race, and histology. Quantitative HCV-RNA testing was performed at hours 0, 6, 12, 24, 48, and 72; days 7, 10, 14, 28, and 56; and weeks 12, 24, 48, and 72. Twelve HCV/HIV-co-infected and 15 HCV-monoinfected patients underwent viral kinetic sampling. Among HIV-positive patients the mean CD4(+) count was 325 cells/mm(3). Seventy-five percent of patients were genotype 1. The HCV-RNA level was undetectable at 72 weeks in 25% and 40% of co-infected and monoinfected patients, respectively. Phase 1/2 declines, free virus clearance rate, and infected hepatocyte death rate were not affected by co-infection status but differed by treatment. Efficiency (epsilon) > or = 90% at 60 hours was associated with viral clearance ( P = .02). Modeling with pooled parameters suggests baseline viral load is a key factor in time to response in this cohort. Predicted clearance time increased by 28% in co-infected patients. Co-infection status did not affect key kinetic parameters. Among kinetic parameters, efficiency was associated significantly with viral clearance. Co-infected patients may require longer treatment duration than monoinfected patients given their generally higher baseline viral loads.

Liquid chromatography/tandem mass spectrometry for the determination of carbamazepine and its main metabolite in rat plasma utilizing an automated blood sampling system

A liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for the simultaneous determination of carbamazepine and its main metabolite carbamazepine 10,11-epoxide in rat plasma is described. The method consists of a liquid–liquid extraction procedure and electrospray LC/MS/MS analysis. The chromatographic separation was achieved within 5 min using a C 8 (150 mm × 2.1 mm) 5 μm column with a mobile phase composed of water/acetonitrile/acetic acid (69.5:30:0.5, v/v/v) at a flow rate of 0.4 ml/min. D 10-carbamazepine is used as the internal standard for all compounds. Analytes were determined by electrospray ionization tandem mass spectrometry in the positive ion mode using selected reaction monitoring (SRM). Carbamazepine was monitored by scanning m/ z 237 → 194, carbamazepine 10,11-epoxide by m/ z 253 → 210 and d 10-carbamazepine by m/ z 247 → 204. The lower limit of quantitation (LLOQ) is 5 ng/ml for each analyte, based on 0.1 ml aliquots of rat plasma. The extraction recovery of analytes from rat plasma was over 87%. Intra-day and inter-day assay coefficients of variations were in the range of 2.6–9.5 and 4.0–9.6%, respectively. Linearity is observed over the range of 5–2000 ng/ml. This method was used for pharmacokinetic studies of carbamazepine and carbamazepine 10,11-epoxide in response to two different blood sampling techniques (i.e., manual sampling versus automated sampling) in the rat. Several differences between the two sampling techniques suggest that the method of blood collection needs to be considered in the evaluation of pharmacokinetic data.