Sunil S. Iyer

Evaluation of the Effect of Ammonia on Nicotine Pharmacokinetics Using Rapid Arterial Sampling

INTRODUCTION The nicotine bolus theory states that the dependence-producing potential of cigarettes relates to a rapid increase in nicotine at brain receptor sites. It has been suggested that ammonia, a compound typically found in tobacco products, further increases the amount of nicotine absorbed and its absorption rate. The aim of this study was to determine whether different ammonia yields in cigarettes affected the rate or amount of nicotine absorption from the lungs to arterial circulation. METHODS 34 adult smokers received 3 separate puffs from each of 2 test cigarettes with different ammonia yields (ammonia in smoke: 10.1 μg per cigarette vs. 18.9 μg per cigarette), followed by rapid radial arterial blood sampling (maximum one sample per second) with 30 min between puffs. Arterial blood samples were assayed for nicotine by liquid chromatography tandem mass spectrometry. Pharmacokinetic modeling was performed and the two test cigarettes were assessed for bioequivalence. RESULTS No significant differences were found in area under the curve, C(max), or T((max)) and the 2 test cigarettes were found to be bioequivalent based on 2 one-sided tests at a significance level of 5%. In addition, the zero-order rate constant (k(0)) obtained from the initial slope of the curves and the model-dependent first-order rate constant (k(a)) were not significantly different. CONCLUSIONS This study provides strong evidence that the different ammonia yields of the test cigarettes had no impact on nicotine pharmacokinetics; thus, the ammonia did not increase the rate or amount of nicotine absorption from a puff of cigarette smoke.

A Validated High-Performance Liquid Chromatography-Tandem Mass Spectrometric (Lc-Ms/Ms) Method for Simultaneous Determination of R(

We report a selective, accurate, and reproducible liquid chromatography-tandem mass spectrometric (LC-MS/MS) method that employs solid phase extraction for quantification of ketorolac enantiomers in human plasma. Resolution of R(

Quantification of Cyclosporin A in Human Cerebrospinal Fluid by Liquid Chromatography‐Mass Spectrometry using Atmospheric Pressure Chemical Ionization

Abstract Cyclosporin A (CsA) is widely used as an immunosuppressant in organ transplantation, and the treatment of autoimmune disorders. This is the first publication describing a rapid, sensitive, and selective liquid chromatography‐mass spectrometry (LC‐MS) method for analysis of CsA in cerebrospinal fluid (CSF). Chromatographic separation was achieved using a reversed‐phase column with a linear gradient mobile phase. A column switching procedure was incorporated to minimize contamination in the ion source of the mass spectrometer. The sample volume requirement was 50 µL and no internal standard was employed. Quantification was carried out with selected ion reaction (SIR) monitoring of the protonated sodium ion adduct (m/z=1224.7). Calibration curves were linear from 0.5 ng/mL to 20 ng/mL with a weighting factor of 1/x. Precision and accuracy, and reverse predicted concentration residuals were within 15%. The method has been used successfully for the analysis of CSF samples from a clinical study.

Rapid automated blood sampling system for pharmacokinetics studies of cigarette smoking.

INTRODUCTION We developed an automated sampling system to allow multiple, discrete blood samples from a human participant to be collected rapidly and immediately following cigarette smoke exposure. We reported the details of the sampling system along with the results of a pilot study for evaluation of the system. METHODS Components of the system include silastic tubing, solenoid pinch valves, a peristaltic pump, and a fraction collector. This system incorporates a smoking machine that allows precise delivery of cigarette smoke through a mouthpiece and intricate timing to correlate blood samples with smoke inhalation. All components are controlled via integration from a user interface and are fully customizable. We performed several tests to evaluate the equipment, including tubing dead volume, leakage tests, and sample reproducibility. We also performed a pilot study with 6 adult smokers, who received 6 controlled puffs of a research test cigarette. Each inhalation was followed by radial arterial blood collection (1 sample per second tapered to 1 sample every 4 s) for 1 min. Samples were evaluated for nicotine via liquid chromatography-tandem mass spectrometric methods. RESULTS Sampling times and volumes were sufficient for nicotine analysis. No adverse effects were seen in the pilot study, and a 30-min washout period was deemed appropriate between puffs. A significant rise in plasma nicotine levels above baseline after inhalation of smoke was consistently detected in all participants. DISCUSSION The unique advantage of this system is to allow rapid blood sampling after a puff of cigarette smoke, with the benefits of reproducibility, reduction in labor intensity, and high temporal resolution.

Development and validation of a LC-ESI-MS/MS method for simultaneous quantification of olmesartan and hydrochlothiazide in human K3 EDTA plasma and its application to pharmacokinetic biostudy

Abstract This is the first publication on a complete validated bioanalytical method for estimation of olmesartan (OLM) and hydrochlorothiazide (HCTZ) in human K3 EDTA plasma that chromatographically resolves its olmesartan glucuronide. An API 4000 mass spectrometer was employed in this method where olmesartan d4 (OLMD4) and hydrochlorothiazide −13C, d2 (HCTZD2) served as the internal standard. Sample was prepared by solid phase extraction (SPE) technique using a polymer based, MCX cartridges and chromatographic resolution achieved on Synergi MAX RP-18A, (4.6 × 150 mm, 4 μm) column using a mobile phase of 0.2% formic acid solution/acetonitrile (30:70, v/v). Negative mass transitions (m/z) of OLM, HCTZ, OLMD4, and HCTZD2 were detected in multiple reactions monitoring (MRM) mode at 445.5 → 149.3, 296.0 → 269.0, 449.2 → 149.3, and 299.1→270.0, respectively. The linearity was checked over a concentration range of 4.051–2500.912 ng/mL for OLM and 0.506–304.109 ng/mL for HCTZ. Intra- and inter-run precision of OLM and HCTZ assay at four concentration levels were below 3.7% and 4.3%, and accuracy was within ±4.4% and 3.0%, respectively. Mean recoveries for OLM, HCTZ, and internal standards OLMD4 and HCTZD2 were 75.68, 77.60%, and 80.2, 89.1%, respectively. This method has been successfully applied to pharmacokinetic biostudy.

Development and validation of a LC-ESI-MS/MS method in human plasma for quantification of fenofibric acid, involving chromatographic resolution of fenofibric acid acyl-β-D-glucuronide

This is the first publication on a validated bioanalytical method for estimation of fenofibric acid in human EDTA plasma that chromatographically resolves its acyl glucuronide. An API 3000 mass spectrometer was employed in this method, and fenofibric acid-d6 served as the internal standard. Sample preparation involved solid phase extraction (SPE) using a polymer based, hydrophilic-lipophilic balanced cartridges and samples were chromatographed on a Discovery C18, (4.6 × 50 mm, 5 μm) column using a mobile phase of 0.2% formic acid solution/acetonitrile (35 : 65, v/v). Negative mass transitions (m/z) of fenofibric acid and fenofibric acid-d6 were detected in multiple reactions monitoring (MRM) mode at 317.1 → 230.9 and 322.9 → 230.8, respectively. The method was validated over a concentration range of 0.150 μg mL−1 to 20.383 μg mL−1. Intra- and inter-run imprecision of fenofibric acid assay at four concentration levels was below 2.5%, and inaccuracy was within ±2.8%. Analytical recoveries for fenofibric acid and internal standard were 73.8–75.4%, and 85.9%, respectively. All other validation parameters were within acceptable limits. The back conversion of fenofibric acid from its acyl glucuronide was minimized by processing samples in an ice cold water bath under low light.

A Multi-centric Bioequivalence Trial in Ph+ Chronic Myeloid Leukemia Patients to Assess Bioequivalence and Safety Evaluation of Generic Imatinib Mesylate 400 mg Tablets

Purpose This study was designed to characterize the pharmacokinetic profile and to assess bioequivalence of the sponsor’s test formulation (imatinib mesylate 400 mg tablets) with an innovator product (Gleevec 400 mg tablets, Novartis Pharmaceuticals) under fed conditions, in adult patients of Philadelphia chromosome positive chronic myeloid leukemia (Ph+ CML) stabilized on imatinib mesylate 400 mg. In addition, the aim of this study was to monitor the safety profile of investigational medicinal products (IMPs). Materials and Methods A multicenter, randomized, open label, two-period, crossover, single dose bioequivalence study was designed for conduct under fed conditions in 42 adult Ph+ CML patients already stabilized on imatinib 400 mg tablets. Pharmacokinetic parameters Tmax, Cmax, and AUC0-24 were calculated using a non-compartmental model on validated WinNonlin software. Validated SAS software was used for statistical evaluation of data. The safety profile of investigational products was monitored during the course of study by applying a clinical process for recording observed untoward effects postadministration of investigational products. Results The 90% confidence intervals for the test/reference mean ratios of the ln-transformed PK variables Cmax (99.0%) and AUC0-24 (99.2%) were within an acceptable range of 80%-125%, as per bioequivalence assumptions. Both formulations were well tolerated after oral administration of IMPs. Conclusion The test product was found to be bioequivalent and safe, and thus can be used interchangeably in clinical practice.

CHALLENGES IN BIOANALYTICAL METHOD DEVELOPMENT FOR SIMULTANEOUS DETERMINATION OF OLMESARTAN AND HYDROCHLOROTHIAZIDE IN HUMAN PLASMA BY LIQUID CHROMATOGRAPHY COUPLED TO TANDEM MASS SPECTROMETRY

A bioanalytical method for simultaneous estimation of olmesartan (OLM) and hydrochlorothiazide (HCTZ) in human K3 EDTA plasma is described. An API 3000 mass spectrometer was employed in this method where olmesartan d4 (OLMD4) and hydroflumethiazide (HFTZ) served as the internal standard. Sample preparation involved solid phase extraction (SPE), a polymer based, hydrophilic-lipophilic balanced cartridges, and chromatographic resolution achieved on X Terra RP18, (4.6 × 150 mm, 5 µm) column using a mobile phase of 2 mM ammonium formate buffer, (pH 3.50 ± 0.10 with formic acid)/acetonitrile (30:70, v/v). Negative mass transitions (m/z) of OLM, HCTZ, OLMD4, and HFTZ were detected in multiple reactions monitoring (MRM) mode at 445.6 → 148.4, 295.9 → 268.9, 449.4 → 148.4, and 329.9 → 238.3, respectively. The linearity was checked over a concentration range of 0.11 × 101 to 1.06 × 103 ng/mL for OLM and 0.10 × 101 to 0.32 × 103 ng/mL for HCTZ. Intra- and inter-run precision of OLM and HCTZ assay at four concentration levels were below 8.1% and 8.0%, and accuracy was within ±3.6% and 9.0%, respectively. Recoveries for OLM, HCTZ, and internal standards OLMD4 and HFTZ were 70.4, 63.5%, and 97.1, 75.2%, respectively. The pre-validation exercises for OLM/HCTZ in human plasma were done using special conditions such as ice cold water bath under low light.

Determination of pharmacokinetics of itraconazole in healthy Indian subjects under fed condition and incurred sample analysis using a validated liquid chromatography tandem mass spectrometric method

This study aimed to determine Itraconazole Pharmacokinetics in healthy Indian subjects under fed condition and incurred sample analysis in human plasma by using a validated liquid chromatography tandem mass spectrometric method. A study in healthy human Indian subjects under fed condition was conducted to determine pharmacokinetics of two treatments of Itraconazole 100 mg capsules. Method validation was performed as per USFDA guidelines. Incurred Sample Reanalysis (ISR) was performed by random selection of subject samples. All the method validation parameters were found to be acceptable in terms of accuracy and precision. Results of all stability exercises in human plasma were within acceptable limits. The validated LCMS/MS method was used for sample analysis. A value of 96.7% for Itraconazole and 92.2% for Hydroxy Itraconazole was observed for incurred sample re-analysis. Arithmetic means for Cmax and AUC0→∞ were 103.699, 95.105 ng/ml and 1665.599, 1556.486 ng/ml.h, respectively, for Itraconazole and for Hydroxy itraconazole Cmax and AUC0→∞ were 179.436, 173.037 ng/ml and 4136.456, 4049.746 ng/ml.h, respectively, in treatment 1 and 2. In conclusion, a LCMS/MS method for quantification of Itraconazole and Hydroxy Itraconazole was used for analysis of clinical samples and determination of pharmacokinetic of Itraconazole in a fed study in an Indian population. Incurred sample re-analysis data indicate good acceptance and reproducibility of the method.

A pharmacokinetic drug interaction study of ceftazidime with clavulanic acid in healthy male Indian subjects

Ceftazidime is a third generation cephalosporin with a broad range of activity. Clavulanic acid is a β-lactamase inhibitor. A fixed dose combination, with the wide spectrum of action of ceftazidime and clavulanic acid’s high stability to β-lactamases has been developed by Ranbaxy Laboratories Limited (India). The present study was planned to predict any interaction between ceftazidime and clavulanic acid which could affect the safety and efficacy of the fixed dose combination. The study was an open label, balanced, randomized two-treatment, two-period, two-sequence, crossover, single-dose comparative pharmacokinetic study under fed conditions. A single intravenous injection of the test product containing a fixed dose combination of ceftazidime 2000 mg and potassium clavulanate 200 mg and the reference product containing ceftazidime 2000 mg only, was administered during each period. Serial blood samples were collected until 12 h post-dose in each period. Ceftazidime and clavulanic acid concentration in plasma were determined using two separate LC-MS/MS methods and then pharmacokinetic parameters were evaluated. No significant difference was seen in the mean Tmax, Cmax, AUC0–t, and AUC0–∞ when the drug was administered as ceftazidime alone and in combination with clavulanic acid. The Confidence Intervals for the log transformed parameters Cmax, AUC0–t and AUC0–∞ were within limits of 80–125% of ceftazidime for the test and reference products. The lack of significant difference between the pharmacokinetic parameters for ceftazidime alone and in the presence of clavulanic acid ruled out any significant interaction between ceftazidime and clavulanic acid.