Determination of hexahydrophthalic acid and methylhexahydrophthalic acid in plasma after derivatisation with pentafluorobenzyl bromide using gas chromatography and mass spectrometric detection

A method for the simultaneous determination of hexahydrophthalic acid (HHP acid) and methylhexahydrophthalic acid (MHHP acid) in human plasma was developed. The procedure was a rapid, single step extractive derivatisation with pentafluorobenzyl bromide as the derivatisation agent. The formed pentafluorobenzyl esters were analysed by gas chromatography-mass spectrometry in negative ion chemical ionisation mode with ammonia as the moderating gas. Deuterium-labeled HHP acid and MHHP acid were used as internal standards. The detection limit was 0.4 ng/ml for HHP acid (m/z 153) and 0.3 ng/ml for MHHP acid (m/z 365). The within-day precision of the method was between 2 and 3% and the between-day precision was between 3 and 12%. The overall recovery was between 65 and 83%. A comparison between HHP acid determinations with a previous and this method showed that the methods gave similar results. The method was applicable for analysis of plasma from occupationally exposed workers.     

Gas chromatographic determination of low concentrations of benzoic acid in human plasma and urine

A method for the determination of benzoic acid down to concentrations of 10 ng/ml in plasma or urine is described. After addition of an internal standard, benzoic acid is extracted at acid pH into diethyl ether. Both compounds are derivatized with pentafluorobenzyl bromide. The derivatives are determined by gas chromatography using a ⁴³Ni electron-capture detector. Hippuric acid is hydrolysed in plasma and urine and total benzoic acid is determined by the same technique.     

Gas chromatographic determination of methamphetamine and its metabolite amphetamine in human plasma and urine following conversion to N-propyl derivatives

A gas chromatographic method for the simultaneous determination of methamphetamine and its metabolite amphetamine in human plasma and urine is described. The method utilizes reductive alkylation with propionaldehyde and sodium borohydride to produce N-propyl derivatives, which have excellent chromatographic properties. Structural analogs of the analytes, p-methylmethamphetamine and p-methylamphetamine, are used as internal standards. The method has good precision and accuracy for concentrations ranging from less than 10 ng/ml to 5000 ng/ml and has been used to measure plasma concentrations as part of a pharmacokinetic/pharmacodynamic study of methamphetamine in humans.     

Determination of dioxopiperazine metabolites of quinapril in biological fluids by gas chromatography—mass spectrometry

The dioxopiperazine metabolites of quinapril in plasma and urine were extracted with hexane—dichloroethane (1:1) under acidic conditions. Following derivatization with pentafluorobenzyl bromide and purification of the desired reaction products using a column packed with silica gel, the metabolites were analysed separately by capillary column gas chromatography—electron-impact mass spectrometry with selected-ion monitoring. The limits of quantitation for the metabolites were 0.2 ng/ml in plasma and 1 ng/ml in urine. The limits of detection were 0.1 ng/ml in plasma and 0.5 ng/ml in urine, at a signal-to-noise ratio of > 3 and > 5, respectively. The proposed method is applicable to pharmacokinetic studies.     

Quantitative analysis of exogenous peptides in plasma using immobilized enzyme cleavage and gas chromatography-mass spectrometry with negative ion chemical ionization

A method is presented for the analysis of peptides in plasma at picomole to femtomole levels. Peptides are isolated from plasma by solid-phase extraction, the peptide of interest is purified by reversed-phase high-performance liquid chromatography (HPLC) and selectively digested using immobilized trypsin or chymotrypsin to yield specific di- or tripeptides. These di- and tripeptides are esterified using heptafluorobutyric anhydride, alkylated with pentafluorobenzyl bromide, then quantified by gas chromatography-mass spectrometry with negative ion chemical ionization. This method has been evaluated for a model synthetic heptapeptide, using a deuterium labeled analog as an internal standard. The half-life of the heptapeptide in human plasma was found to be 2 min. Extraction efficiencies of a tritiated peptide of similar size to the heptapeptide, [³H]DSLET, from plasma using either C₁₈ or strong cation-exchange columns were 85±3 and 70±2%, respectively. Quantitation of fragments from the heptapeptide indicated that the analysis was linear from 1–50 ng of the heptapeptide per ml of plasma. This method was subsequently employed for pharmacokinetic studies of the biologically active peptide Met-enkephalin-Arg-Gly-Leu, where linearity was obtained from 50 to 1000 ng/ml in rat plasma. This method demonstrated negligible side reaction by-products due to autolysis, and has potential for extensive use given the wide availability of gas chromatography-mass spectrometry.     

Simultaneous determination of plasma levels of fluvoxamine and of the enantiomers of fluoxetine and norfluoxetine by gas chromatography-mass spectrometry

A gas chromatographic-mass spectrometric method is presented which allows the simultaneous determination of the plasma concentrations of fluvoxamine and of the enantiomers of fluoxetine and norfluoxetine after derivatization with the chiral reagent, (S)-(-)-N-trifluoroacetylprolyl chloride. No interference was observed from endogenous compounds following the extraction of plasma samples from six different human subjects. The standard curves were linear over a working range of 10 to 750 ng/ml for racemic fluoxetine and norfluoxetine and of 50 to 500 ng/ml for fluvoxamine. Recoveries ranged from 50 to 66% for the three compounds. Intra- and inter-day coefficients of variation ranged from 4 to 10% for fluvoxamine and from 4 to 13% for fluoxetine and norfluoxetine. The limits of quantitation of the method were found to be 2 ng/ml for fluvoxamine and 1 ng/ml for the (R)- and (S)-enantiomers of fluoxetine and norfluoxetine, hence allowing its use for single dose pharmacokinetics. Finally, by using a steeper gradient of temperature, much shorter analysis times are obtained if one is interested in the concentrations of fluvoxamine alone.     

Determination of methimazole in plasma using gas chromatography—mass spectrometry after extractive alkylation

A gas chromatography—mass spectrometry method for quantitation of the thyreostatic agent methimazole in plasma is described. The drug was transferred from the plasma sample and derivatized in one step by extractive alkylation. Either of two alkylating agents benzylchloride or pentafluorobenzyl bromide were used. Deuterium-labelled methimazole was used as internal standard. The precision of the method at the level of 5 ng methimazole per ml plasma was 6%.     

A high-performance liquid chromatography assay with ultraviolet detection for olanzapine in human plasma and urine

Olanzapine is a commonly used atypical antipsychotic medication for which therapeutic drug monitoring has been proposed as clinically useful. A sensitive method was developed for the determination of olanzapine concentrations in plasma and urine by high-performance liquid chromatography with low-wavelength ultraviolet absorption detection (214 nm). A single-step liquid–liquid extraction procedure using heptane-iso-amyl alcohol (97.5:2.5 v/v) was employed to recover olanzapine and the internal standard (a 2-ethylated olanzapine derivative) from the biological matrices which were adjusted to pH 10 with 1 M carbonate buffer. Detector response was linear from 1–5000 ng (r²>0.98). The limit of detection of the assay (signal:noise=3:1) and the lower limit of quantitation were 0.75 ng and 1 ng/ml of olanzapine, respectively. Interday variation for olanzapine 50 ng/ml in plasma and urine was 5.2% and 7.1% (n=5), respectively, and 9.5 and 12.3% at 1 ng/ml (n=5). Intraday variation for olanzapine 50 ng/ml in plasma and urine was 8.1% and 9.6% (n=15), respectively, and 14.2 and 17.1% at 1 ng/ml (n=15). The recoveries of olanzapine (50 ng/ml) and the internal standard were 83±6 and 92±6% in plasma, respectively, and 79±7 and 89±7% in urine, respectively. Accuracy was 96% and 93% at 50 and 1 ng/ml, respectively. The applicability of the assay was demonstrated by determining plasma concentrations of olanzapine in a healthy male volunteer for 48 h following a single oral dose of 5 mg olanzapine. This method is suitable for studying olanzapine disposition in single or multiple-dose pharmacokinetic studies.     

Gas chromatographic–tandem mass spectrometric determination of acetylsalicylic acid in human plasma after oral administration of low-dose aspirin and guaimesal

A fully validated gas chromatographic–tandem mass spectrometric (GC–MS–MS) method is described for the accurate determination of acetylsalicylic acid (ASA) in human plasma after a single low-dose oral administration of aspirin or guaimesal, an ASA releasing prodrug. ASA and the newly prepared O-[²H₃]-acetylsalicylic acid (d3-ASA) used as internal standard were determined in 100-μl aliquots of plasma by extractive pentafluorobenzyl (PFB) esterification using PFB bromide and tetrabutylammoniumhydrogen sulphate as the esterifying and ion-pairing agent, respectively, and by GC–MS–MS analysis in the negative-ion chemical ionization mode. The overall relative standard deviations were below 8% for ASA levels in the range 0–1 μg/ml plasma. Mean accuracy was 3.8% for ASA levels within the range 0–100 ng/ml. The limit of quantitation of the method was determined as 200 pg/ml ASA at an accuracy of 5.5% and a precision of 15.2%. The limit of detection was determined as 546 amol of ASA at a signal-to-noise ratio of 10:1.     

Gas chromatographic—mass spectrometric determination of tiopronin in human blood using acrylic acid esters as a derivatization reagent for the thiol group

A gas chromatographic—mass spectrometric method for determining tiopronin, which has a thiol group, in human blood has been described. To prevent the oxidative degradation of tiopronin in the blood, its thiol group was immediately protected by treatment with isobutyl acrylate, which reacted readily with tiopronin in a 0.1 M Na₂HPO₄ solution (pH 9.1). The reaction was quantitative within 30 min. The blood sample was deproteinized and purified by a combination of liquid—liquid extraction and solid-phase extraction. Finally, the carboxyl moiety of the ester adduct was derivatized to the pentafluorobenzyl ester. The derivatives of tiopronin and the internal standard were analysed with gas chromatography—mass spectrometry. The precision of the method was satisfactory, and the calibration curve had good linearity in the concentration range investigated. The limit of determination of tiopronin in blood was estimated to be ca. 1 ng/ml.     

Micro-quantitative determination of ciprostene in plasma by gas chromatography—mass spectrometry coupled with an antibody extraction

A simple and highly sensitive method has been developed for the determination in plasma of ciprostene, 9β-methyl-6α-carbaprostaglandin I₂, using gas chromatography—mass spectrometry following solid-phase extraction on an immobilized antibody column. The anti-ciprostene antibody obtained from rabbit serum was coupled to an agarose support matrix, and the immobilized antibody thus prepared was used as an extraction phase for sample clean-up. The extracted drug was treated with pentafluorobenzyl bromide followed by bis(trimethylsilyl)trifluoroacetamide. The derivative was quantitatively analysed by negative-ion chemical ionization gas chromatography—mass spectrometry. The lower limit of quantitation was 50 pg/ml when 1 ml of human plasma was used. The plasma concentration of ciprostene in a dog treated with ciprostene at 2.5 μg/kg was determined successfully by this method.     

Gas chromatographic determination of phentolamine (regitine®) in human plasma and urine

A method for the determination of unconjugated phentolamine at concentrations down to 6 ng/ml in human plasma, and of free and total (free plus conjugated) phentolamine down to 25 ng/ml in urine is described. After addition of 2-[N-(p-chlorophenyl)-N-(m-hydroxyphenyl)-aminomethyl]-2-imidazoline as internal standard, both compounds are extracted into benzene—ethyl acetate (1:1, v/v) at pH 10, transferred into an acidic aqueous solution and back-extracted at pH 10 into benzene—ethyl acetate. They are then derivatized with N-heptafluorobutyrylimidazole. The derivatives are determined by gas chromatography using a ⁶³Ni electron-capture detector. In urine, total (free plus conjugated) phentolamine is determined after enzymatic hydrolysis. The technique was applied for the study of the plasma concentrations and urinary elimination after oral administration to man.     

Validation of a high-performance liquid chromatography method for tramadol and o-desmethyltramadol in human plasma using solid-phase extraction

An HPLC system using solid-phase extraction and HPLC with UV detection has been validated in order to determine tramadol and o-desmethyltramadol (M1) concentrations in human plasma. The method developed was selective and linear for concentrations ranging from 50 to 3500 ng/ml (tramadol) and 50 to 500 ng/ml (M1) with mean recoveries of 94.36±12.53% and 93.52±7.88%, respectively. Limit of quantitation (LOQ) was 50 ng/ml. For tramadol, the intra-day accuracy ranged from 95.48 to 114.64% and the inter-day accuracy, 97.21 to 103.24%. Good precision (0.51 and 18.32% for intra- and inter-day, respectively) was obtained at LOQ. The system has been applied to determine tramadol concentrations in human plasma samples for a pharmacokinetic study.     

Sensitive, high-throughput gas chromatographic–mass spectrometric assay for fluoxetine and norfluoxetine in human plasma and its application to pharmacokinetic studies

A sensitive, robust gas chromatographic–mass spectrometric assay suitable for use in pharmacokinetic or bioequivalence studies is presented for the selective serotonin reuptake inhibitor, fluoxetine, and its major metabolite, norfluoxetine (N-desmethylfluoxetine). This method employs solid-phase extraction followed by acetylation with trifluoroacetic anhydride and analysis of the derivatives using selected ion monitoring. The lower limit of quantification was 1.0 ng/ml, and the assay was linear for both analytes from 1 to 100 ng/ml. Mean recoveries following solid-phase extraction at concentrations of 5.0, 20 and 100 ng/ml were 91% (fluoxetine) and 87% (norfluoxetine). Assay precision (as mean RSD) and accuracy (as mean relative error) for both analytes were tested at the same three nominal concentrations and were found to be within 10% in all cases. Analysis of fluoxetine concentrations in plasma samples from 18 volunteers following administration of a single 40 mg dose of fluoxetine provided the following pharmacokinetic data (mean±SD): C_max, 32.73±9.21 ng/ml; AUC_0–∞, 1627±1372 ng/ml h; T_max, 3.08 h (median); k_e, 0.022±0.007 h⁻¹; elimination half-life, 37.69±21.70 h.     

Simultaneous determination of norethisterone and six metabolites in human plasma by capillary gas chromatography with mass-selective detection

A method for the simultaneous determination of norethisterone (NET) and six metabolites in human plasma by capillary gas chromatography-mass-selective detection (GC-MS) is described. The compounds are determined in plasma after enzymatic hydrolysis. After addition of norgestrel as the internal standard, the compounds are extracted from plasma at pH 5 using an Extrelut column and elution with dichloromethane. After evaporation, the compounds are converted into bistrimethylsilyl derivatives which are determined by gas chromatography using a mass-selective detector at m/z 429 for the two dihydro-NET (5β-NET and 5α-NET), m/z 431 for the four tetrahydro-NET (3α,5α-NET, 3α,5β-NET, 3β,5β-NET and 3β,5α-NET), m/z 442 for NET and m/z 456 for the internal standard. The reproducibility and accuracy of the method were found suitable over the range of concentrations between 0.50 and 8 ng/ml for NET, and metabolites except for 5α-dihydro-NET (between 1 and 8 ng/ml). The method was applied to clinical samples.     

Determination of flumazenil in plasma by gas chromatography-negative ion chemical ionization mass spectrometry

A gas chromatographic-negative ion chemical ionization mass spectrometric (GC-NCI-MS) method for the determination of flumazenil in plasma is described. The GC of flumazenil (M_r 303) is considered to be difficult as it is readily adsorbed in the GC column. Therefore, preconditioning the GC column with reconstituted extract from plasma and Silyl-8 was required to cover the active sites on the column. Monitoring the maximum mass peak (m/z 275) of the flumazenil resulted in a tenfold enhancement of sensitivity and signal-to-noise ratio (concentration = 1 ng/ml). Isotopically labeled flumazenil-d₃ (M_r 306, m/z 278) was used as the internal standard. The detection limit for flumazenil was found to be 0.1 ng/ml with an injection volume of 2 μl. The signal-to-noise ratio was about 10. The routine quantification limit was set at 2 ng/ml for dog plasma and 1 ng/ml for human plasma. The sample volumes in both instances were 1 ml.     

Evaluation and routine application of the novel restricted-access precolumn packing material Alkyl-Diol Silica: coupled-column high-performance liquid chromatographic analysis of the photoreactive drug 8-methoxypsoralen in plasma

A fully automated coupled-column HPLC method for on-line sample processing and determination of the photoreactive drug 8-methoxypsoralen (8-MOP) in plasma has been developed. The method is based on the novel internal-surface reversed-phase precolumn packing materials Alkyl-Diol Silica (ADS). This new family of restricted-access materials has a hydrophilic, electroneutral outer particle surface and a hydrophobic internal pore surface. The supports tolerate the direct and repetitive injection of proteinaceous fluids such as plasma and allow a classical C₁₈-, C₈- or C₄-reversed-phase partitioning at the internal (pore) surface. The total protein load, i.e. the lifetime of the precolumn used in this study (C₈-Alkyl-Diol Silica, 25 μm, 25 × 4 mm I.D.), exceeds more than 100 ml of plasma. 8-MOP was detected by its native fluorescence (excitation 312 nm, emission 540 nm). Validation of the method revealed a quantitative and matrix-independent recovery (99.5–101.3% measured at five concentrations between 21.3 and 625.2 ng of 8-MOP per milliliter of plasma), linearity over a wide range of 8-MOP concentrations (1.2–3070 ng of 8-MOP/ml, r = 0.999), low limits of detection (0.39 ng of 8-MOP/ml) and quantitation (0.79 ng of 8-MOP/ml) and a high between-run (C.V. 1.47%, n = 10) and within-run (C.V. 1.33%, n = 10) reproductivity. This paper introduces coupled-column HPLC as a suitable method for on-site analysis of drug plasma profiles (bedside-monitoring).     

Improved method for routine determination of nicotine and its main metabolites in biological fluids

Extrelut^R extraction and glass capillary gas chromatography were applied to the routine determination of nicotine and its metabolites cotinine, nicotine-1′-N-oxide and cotinine-1-N-oxide in urine and plasma. After extraction of nicotine and cotinine both N-oxides and phendimetrazine-N-oxide (used as internal standard) were reduced to their bases by SO₂ on-column and eluted by a mixture of diethyl ether and dichloromethane. The minimum detectable concentrations are 0.03 μg/ml for urinary nicotine and cotinine and 0.1 μg/ml for the N-oxides. In plasma samples the corresponding values are 5 ng/ml and 15 ng/ml, respectively, with sample values as small as 2 ml. The advantage of the direct determination of all four compounds of interest in one sample reduced the amount of plasma required. The straightforward and rapid extraction and reduction procedure as well as the long-term stability of the gas chromatographic separation system make the method suitable for routine application.     

High-performance liquid chromatographic assay of propofol in human and rat plasma and fourteen rat tissues using electrochemical detection

This paper describes a sensitive HPLC-electrochemical detection analytical method for determining the concentration of the intravenous anesthetic, propofol, in human or rat plasma or serum and a variety of rat tissues. Internal standard and drug are extracted from serum or plasma and other tissues with pentane. 2,6-tert.-Butylmethylphenol is used as internal standard. It includes a novel steam distillation procedure for separating the highly lipophilic propofol from skin and fat. The plasma/serum assay has a precision of 1–4% (C.V.) in the range 10 ng/ml to 1 μg/ml and permits the assay of 5 ng/ml from 0.1 ml of plasma/serum. The tissue procedure allows the estimation of 50 ng/g in 0.1 g of tissue for most of the major organs with less than 2% (C.V.) precision. This assay was used to measure propofol concentrations in plasma/serum and tissue samples in support of a project to develop a physiological pharmacokinetic model for propofol in the rat.     

Solid-phase microextraction with capillary gas-liquid chromatography and nitrogen-phosphorus selective detection for the assay of antidepressant drugs in human plasma

Solid-phase microextraction (SPME) was tested as a sample preparation for the simultaneous assay of ten antidepressant drugs and metabolites (TADs) in human plasma. Aqueous NaOH (0.5 ml, 1 M) and chloramitriptyline (50 μl, 40 μg/ml) as internal standard (I.S.) were added to a 2-ml plasma sample. This mixture was extracted with a 100-μm polydimethylsiloxane SPME fiber (Supelco) for 10 min. After washing in water and methanol (50%) and subsequent drying at room temperature, desorption of the fiber was performed in the injection port of a gas chromatograph at 260°C for 1 min (HP 5890, DB-17 30 m×0.25 mm I.D., 0.25 μm capillary; 0.7 ml/min nitrogen; nitrogen-phosphorus selective detection). The recovery was found to be very low from plasma (0.3% to 0.8%) but considerably higher from water (about 15%). Therefore, the high protein binding of antidepressants appears to be the main limiting mechanism for a better extraction. However, the analytes were well separated and the calibrations were linear between 125 ng/ml and 2000 ng/ml. The limits of quantification were about 90 ng/ml for imipramine and desipramine, 125 ng/ml for amitriptyline, trimipramine, doxepine, nortriptyline and mianserine and about 200 ng/ml for maprotiline, clomipramine and desmethylclomipramine. The recovery was improved by increasing the extraction time. The influence of the concentrations of the sum of proteins and of α-acid glycoprotein on the peak-area ratios A_TAD/A_I.S. and on absolute peak areas was studied. Peak-area ratios increased with decreasing protein concentration but were found to be independent on α-acid glycoprotein. A simple model for the explanation of the effect is presented. Measures for the improvement of sensitivity are discussed. As presented in this paper, which first describes SPME for the analysis of drugs in plasma, SPME with a short extraction time can be of only very limited value for therapeutic drug monitoring. Lower concentrations than the limit of quantification are usually found at therapeutic doses. The method can be useful for toxicological analysis after the accidental or suicidal intake of higher doses. However, an about 10-fold improvement of the sensitivity of the method seems to be possible.