Specific Gas Chromatography-mass Spectrometry Method Research Articles

The development and validation of a gas chromatography-mass spectrometry method for the determination of metformin in human plasma

A new, simple, specific gas chromatography-mass spectrometry method (GC-MS) was developed for the determination of metformin in human plasma. A number of derivatization approaches (silylation, acylation and methylation) were tested to derivatize metformin prior to GC-MS analysis. Derivatization of metformin was achieved by using N-methyl bis(trifluoroacetamide) (MBTFA). Several parameters such as reaction temperature and time were optimized, which affected the yield of the derivatization reaction. A trifluoroacetyl derivative of metformin was identified and quantified in selected ion monitoring mode (mass-to-charge ratio (m/z): 303). The method was fully validated using parameters such as specificity, carryover, linearity, limits of detection and quantification, precision, accuracy, stability, recovery, robustness and ruggedness. Linearity was demonstrated over the concentration range of 100–3000 ng mL−1 with a coefficient of determination (R2) above 0.996. The limits of quantification and detection were found to be 100 and 40 ng mL−1, respectively. Intra- and inter-day accuracy and precision were within the acceptable limits (<15% for concentration points in the calibration range; <20% for the limit of quantitation). The developed method was successfully applied for the identification and quantification of metformin in the plasma of diabetic patients.

HIGH COGNITIVE DIETARY RESTRAINT IS ASSOCIATED WITH INCREASED CORTISOL EXCRETION IN POSTMENOPAUSAL WOMEN: A COMMENT

To the Editor: I read with great interest the paper of Rideout and colleagues (1) who claimed that ‘‘our finding of higher cortisol excretion in postmenopausal women with high cognitive dietary restraint suggests that high dietary restraint may be a source of stress for generally healthy, normal-weight postmenopausal women’’ (p. 631). A shortcoming may confound their work, making the interpretation of their endocrine results difficult, if not impossible. The shortcoming is: The authors measured urinary ‘‘cortisol’’ (probably: urinary free ‘‘cortisol’’) by a competitive chemiluminescent immunoassay (Bayer ADVIA Centaur) that lacks specificity. Gray and colleagues (2) had evaluated this immunoassay and compared their values of urinary free cortisol (UFF) with those obtained by a specific gas chromatography–mass spectrometry method. They observed that cortisone, a major metabolite of UFF in the urine, demonstrated a crossreactivity of 44%, and they concluded that this method overestimates urinary cortisol, giving results up to twice those obtained by a specific gas chromatography–mass spectrometry method. Thus, we may assume that Rideout

Further evidence for the presence of GHB in postmortem biological fluid: implications for the interpretation of findings.

Analysis and interpretation of the findings for the drug of abuse gamma hydroxybutyric acid (GHB) in fatalities has become very problematic. This is primarily because of variable data in postmortem biological fluids resulting from the endogenous nature of the compound, possible postmortem production, and varying methods of detection. Preliminary studies support the use of plasma standards in determining urinary GHB concentrations and indicate measurement of GHB in postmortem biological fluids may be dependent on the method of analysis. In order to assist interpretation of postmortem data based on gas chromatography-mass spectrometry (GC-MS) analysis using GHB-d6 internal standard, the results of GHB concentrations measured routinely in postmortem blood and urine specimens in 40 fatalities received during a three-month period are shown. In all cases, GHB was not implicated in the cause of death; there was no apparent correlation between manner of death and resultant GHB concentrations. Mean concentrations of GHB determined in postmortem blood were found to be 12.3 mg/L (range = 2-29 mg/L, n = 38) and 12.6 mg/L (range = 4-25 mg/L, n = 17) (unpreserved and sodium fluoride-preserved samples, respectively) and 5.5 mg/L in unpreserved urine (range 0-18 mg/L, n = 39) and 4.8 mg/L in sodium fluoride-preserved (range 0-10 mg/L, n = 15) urine samples. Vitreous humor was available in two of the cases analyzed (GHB = 1 and 3 mg/L). The data support the potential use of sodium fluoride-preserved samples for interpretation of GHB concentrations, particularly if there has been an extended postmortem interval. In addition, interpretative cut-offs can be proposed for both postmortem blood and urine, based on the specific GC-MS method used. At blood concentrations less than 30 mg/L and at urine concentrations less than 20 mg/L, it is possible that any GHB detected could represent only endogenous GHB production.

Poor specificity and recovery of urinary free cortisol as determined by the Bayer ADVIA Centaur extraction method.

Immunoassay methods for urinary free cortisol (UFC) lack specificity, and many procedures have not been fully evaluated for routine use. In the current study we evaluated the Bayer ADVIA Centaur extraction UFC immunoassay and compared results to those obtained by a specific gas chromatography-mass spectrometry (GC-MS) method. The Bayer ADVIA Centaur cortisol assay lacked specificity. Cortisone, a steroid present in urine at concentrations similar to those of cortisol, demonstrated a cross-reactivity of 44%. In addition, the choice of matrix used to resuspend steroids after solvent extraction from urine affected recovery. Recovery was improved if the recommended urine reconstruction buffer was replaced by steroid-free serum. Irrespective of the sample matrix used, the Centaur method overestimates UFC, giving results up to twice those obtained by a specific GC-MS method.

Lack of Whole-body Pharmacokinetic Differences of Halothane Enantiomers in the Rat

Halothane is made and used as a racemate (an equimolar mixture of R- and S- enantiomers). This study was initiated to determine whether there were demonstrable enantiomeric differences in the whole-body pharmacokinetics of halothane that might have significance for studies in which racemate is used. Adult male Wistar rats were exposed to halothane vaporized in the atmosphere of a closed constant volume chamber supplied with O2 commensurate with CO2 production. Concentrations of halothane enantiomers were measured by a specific gas chromatography-mass spectrometry method. Experiments were performed at four initial concentrations of halothane (0.1%, 0.5%, 1.0%, and 1.5% vol/vol). Enantiomeric differences in whole-body pharmacokinetics were assessed indirectly from the relative chamber atmosphere concentrations of halothane enantiomers. Concentrations of halothane decreased biphasically. The initial more rapid decrease was interpreted as incorporating absorption, distribution, and clearance; the slower decrease was interpreted as principally incorporating metabolic clearance. The ratio of concentrations of the two halothane enantiomers and of the ratios of the respective areas under the concentration-time curves remained constant without differing from unity at any time at any concentration of halothane. The dose-normalized areas under the concentration-time curves for the concentrations 0.1%, 0.5%, and 1.0% did not differ; that for 1.5% was significantly greater, suggesting nonlinear clearance, but the values did not differ significantly between enantiomers at any concentration. As there were no significant differences in concentrations of the two enantiomers in the chamber atmosphere, enantioselectivity was not demonstrated in the whole-body pharmacokinetics of halothane.