Determination of 2',3'-dideoxycytidine in maternal plasma, amniotic fluid, placental and fetal tissues by high-performance liquid chromatography.

2',3'-Dideoxycytidine (DDC) is a nucleoside reverse transcriptase inhibitor that has been shown to inhibit the human immunodeficiency virus (HIV). DDC is a candidate for treatment of pregnant women to prevent prenatal transmission of HIV/AIDS to their unborn children. A quick and simple high-performance liquid chromatography (HPLC) method has been developed and validated for the determination of DDC concentrations in samples collected from a pregnant rat model (maternal plasma, amniotic fluid, placental and fetal tissues). Extraction of DDC and its internal standard 2',3'-dideoxy-3'-thiacytidine (3TC) in plasma and amniotic fluid was carried out by protein precipitation. Extraction from placental and fetal homogenates was achieved by solid phase extraction using Waters Oasis HLB solid phase extraction cartridges. Chromatographic separation was achieved on a Waters Spherisorb S3W silica column (4.6 mm x 100 mm) equipped with a Phenomenex guard column. The mobile phase used was 10% methanol in water with 22 mM formic acid. The flow rate was 0.5 ml/min, and the detection wavelength was optimized at 275 nm. Under these chromatographic conditions, DDC eluted around 12 min, and 3TC eluted around 10 min. The calibration curves for each day of validation and analysis showed good linear response through the range of 0.15-75.0 microg/ml in each of the four matrices. The relative recovery for DDC in each of the matrices ranged from 87.8% to 103.0%. Acceptable intra- and inter-day assay precision (<15% R.S.D.) and accuracy (<15% error) were observed over 0.15-75.0 microg/ml for all four matrices.

Common bioanalytical deficiencies with bioequivalence submissions in Abbreviated New Drug Applications.

BACKGROUND: The US FDA published A Guidance for Industry: Bioanalytical Method Validation in May 2001. Despite the publication of the guidance, companies continue to submit bioequivalence studies with bioanalytical deficiencies that preclude Abbreviated New Drug Application approval. The Divisions of Bioequivalence in the FDA's Office of Generic Drugs conducted a survey of the bioequivalence submissions over a 10-year period (2001-2011) to identify the most commonly occurring bioanalytical deficiencies. RESULTS: Data from a total of 4028 Abbreviated New Drug Application submissions were collected to identify bioanalytical deficiencies. Of the three categories of bioanalytical deficiencies (method, validation and report), the majority of the deficiencies were from the bioanalytical method validation section. Globally, the percentage of bioanalytical method validation deficiencies was 62%. CONCLUSIONS: The approval of generic drugs would be accelerated if these deficiencies were avoided by generic companies by adhering to the guidance and therefore submitting a more complete application.

Quantitative liquid chromatography/time-of-flight mass spectrometry.

Over the last decade, time-of-flight (TOF) instruments have increasingly been used as quantitation tools. In addition, because of their high resolving power, they can be used for verification of empirical formulas. Historically, TOF instruments have had limited quantitation capabilities because of their narrow dynamic range. However, recent advances have improved these limitations. This review covers the rationale for using TOF for LC detection, and describes the many methods currently in the literature for the quantitation of pharmaceuticals, environmental pollutants, explosives and many phytochemicals.

Quantitative gas chromatography/time-of-flight mass spectrometry: a review.

Time-of-flight (TOF) instruments have recently gained popularity in quantitative analyses. Normally, TOF mass spectrometers are used for accurate mass measurements for empirical formula verification. However, over the past decade, they have been used quantitatively as well. Because of the fast separations and narrow peaks that result from gas chromatography separations, scanning mass spectrometers are not ideal detectors. TOF mass spectrometers, however, have the ability to collect spectra at a faster rate. Two-dimensional gas chromatography has also been introduced to further resolve peaks from complex matrices. Two-dimensional gas chromatography results in a faster separation as well as narrower peaks. This paper reviews the methods currently in the literature for the quantitation of compounds using one- and two-dimensional gas chromatography and TOF mass spectrometry detection.

Chronic, intermittent exposure to chlorpyrifos in rats: protracted effects on axonal transport, neurotrophin receptors, cholinergic markers, and information processing.

Persistent behavioral abnormalities have been commonly associated with acute organophosphate (OP) pesticide poisoning; however, relatively little is known about the consequences of chronic OP exposures that are not associated with acute cholinergic symptoms. In this study, the behavioral and neurochemical effects of chronic, intermittent, and subthreshold exposures to the OP pesticide, chlorpyrifos (CPF), were investigated. Rats were injected with CPF s.c. (dose range, 2.5-18.0 mg/kg) every other day over the course of 30 days and then were given a 2-week CPF-free washout period. In behavioral experiments conducted during the washout period, dose-dependent decrements in a water-maze hidden platform task and a prepulse inhibition procedure were observed, without significant effects on open-field activity, Rotorod performance, grip strength, or a spontaneous novel object recognition task. After washout, levels of CPF and its metabolite 3,5,6-trichloro-2-pyridinol were minimal in plasma and brain; however, cholinesterase inhibition was still detectable. Furthermore, the 18.0 mg/kg dose of CPF was associated with (brain region-dependent) decreases in nerve growth factor receptors and cholinergic proteins including the vesicular acetylcholine transporter, the high-affinity choline transporter, and the alpha(7)-nicotinic acetylcholine receptor. These deficits were accompanied by decreases in anterograde and retrograde axonal transport measured in sciatic nerves ex vivo. Thus, low-level (intermittent) exposure to CPF has persistent effects on neurotrophin receptors and cholinergic proteins, possibly through inhibition of fast axonal transport. Such neurochemical changes may lead to deficits in information processing and cognitive function.

Determination of chlorpyrifos and its metabolites in rat brain tissue using coupled-column liquid chromatography/electrospray ionization tandem mass spectrometry.

A method has been developed to quantify chlorpyrifos (O,O-diethyl-O-[3,5,6,-trichloro-2-pyridyl] phosphorothionate) and its metabolites chlorpyrifos-oxon (O,O-diethyl-O-[3,5,6,trichloro-2-pyridinyl] phosphate) and TCP (3,5,6,-trichloro-2-pyridinol) in rat brain tissue by coupled-column liquid chromatography/electrospray ionization tandem mass spectrometry (LC/LC/ESI-MS/MS). Rat brains were homogenized and treated by protein precipitation using ice-cold acetonitrile. The supernatant was directly injected onto the coupled-column system. Sample clean-up was achieved on a Zorbax Extend-C(18) column (2.1 x 50 mm, 5 microm) using a mobile phase of acetonitrile/water with 0.0025% formic acid (40:60, v/v). The compounds were separated isocratically on a Zorbax Eclipse XDB C(8) column (2.0 x 150 mm, 5 microm) using a mobile phase of acetonitrile/water with 0.0025% formic acid (75:25, v/v). Chlorpyrifos and chlorpyrifos-oxon were detected in positive ion mode using multiple reaction monitoring (MRM). TCP was detected in negative ion mode using precursor-to-precursor transition monitoring. The method was validated and the specificity, linearity, limit of quantitation (LOQ), precision, accuracy, stability, and recoveries were determined. Calibration curves for all three analytes yielded correlation coefficients of 0.993 or greater. The LOQs were 25.3 ng/g for chlorpyrifos and 6.3 ng/g for chlorpyrifos-oxon and TCP. All precision relative standard deviations (RSDs) were less than 16% for the LOQ and less than 11% for the other QC samples. This method was successfully applied to six rats that were injected subcutaneously with chlorpyrifos.