Electron-impact ionization detection of scopolamine by gas chromatography-mass spectrometry in rat plasma and brain

Scopolamine, a muscarinic receptor antagonist, was measured in rat plasma and brain using gas chromatography-mass spectrometry with electron-impact ionization detection. Extracted scopolamine was either directly derivatized or first hydrolyzed to scopine, then derivatized with heptafluorobutyric anhydride, separated on a capillary column and quantified by mass fragmentography. Electron-impact ionization produced a common fragment peak at m/z 138 that was monitored along with trideuterated scopolamine, an internal standard (m/z 141). The method can be used to measure scopolamine concentrations of 2 ng/ml in rat plasma and 20 ng/g in rat brain.     

An HPLC Assay for a Prostacyclin Analogue,Ciprostene Calcium,in Human Plasma

Abstract

A sensitive assay has been developed for the quantification of the prostacyclin analogue, ciprostene calcium, in human plasma. The method involves solid phase extraction of ciprostene calcium and internal standard, carbacyclin, from a small volume of human plasma. The extract is derivatized with 4-bramamethyl-7-acetoxycoumarin, and the derivatized product extracted with a polar solid phase cartridge and concentrated by evaporation. The final extract is separated by reversed phase HPIC and measured by a fluorimetric detector following post-column alkaline hydrolysis. The overall extraction efficiency is better than 75%, and the assay is linear over the concentration range studied (5–20 ng/ml). The limit of quantification was approximately 5 ng/ml. Ultimate sensitivity was limited by interfering peaks endogenous to the biological matrix. Coefficients of variation at mid-range concentrations are less than 10%.     

Square wave anodic stripping voltammetric determination of Pb2+ using acetylene black paste electrode based on the inducing adsorption ability of I-

In the study, we developed a simple, rapid and sensitive method for the determination of tiopronin (TP) in human plasma, which was based on derivatization with p-bromophenacyl bromide (p-BPB) followed by liquid-liquid extraction and reverse-phase HPLC-UV detection. For the first time, the p-BPB was introduced into the derivatization of TP. The thiol group of TP was trapped with p-BPB to form a TP-p-BPB adduct, which can be very suitable for UV detection. From acidified plasma samples, the derivatized TP was extracted with 5 mL dichloromethane. Effective chromatographic separation was achieved using a C18 column (DIAMONSIL 150 mm × 4 mm i.d., 5 μm) based on an acetonitrile-water-trifluoroacetic acid (40:59.88:0.12, v/v/v) elution at a flow-rate of 1 mL/min. The IS and the derivatized TP were detected at 263 nm. No endogenous substances were found to interfere. The limit of quantification for derivatized TP (TP-p-BPB) in plasma was 40 ng/mL. The calibration curve for the derivatized TP showed linearity in the range 0.04-4 μg/mL with a regression coefficient corresponding to 0.9991 and the coefficient of the variation of the points of the calibration curve being lower than 10%. Extraction recoveries of the derivatized TP in plasma were greater than 72%. The method was suitably validated and successfully applied to determination of TP in human plasma samples.     

High-performance liquid chromatographic separation of femtomolar quantities of endogenous carboxylic acids, including arachidonic acid metabolites, as 4-bromomethyl-7-acetoxycoumarin derivatives

A major limitation of high-performance liquid chromatographic techniques for measuring biologically active eicosanoids has been the inadequate sensitivity of most on-line detection systems. In addition, the availability of a technique suitable for measuring small quantities of non-esterified fatty acids (NEFAs) in plasma would allow longitudinal studies of plasma levels of these lipids in small animals. To improve the sensitivity of detection, the compounds with acyl groups containing carboxylic acids were derivatized with the highly fluorescent compound, 4-bromomethyl-7-acetoxycoumarin. All classes of NEFA and arachidonic acid metabolites, including the cyclooxygenase and lipoxygenase products, and hydroxy acid compounds could be derivatized with this reagent. The derivatized metabolites were separated with a reversed-phase high-performance liquid chromatographic system using a radial compression column and a gradient elution technique. Reproducible measurements of plasma NEFAs from as little as 5 microliters of plasma, and femtomolar concentrations of eicosanoids, could be detected using an on-line fluorescent spectrometer. This improvement in sensitivity should permit the quantification of all eicosanoids, including the leukotrienes, in biologic fluids and the longitudinal measurement of changes in plasma NEFA levels in small animals.     

A Sensitive Gas Chromatographic Method for the Determination of Metoprolol in Human Plasma

Abstract

A sensitive method for the determination of metoprolol in plasma has been developed. The procedure is based on gas chromatographic measurements of derivatized metoprolol, using 9-bromophenanthrene as internal standard. Metoprolol is derivatized with pentafluoropropionic anhydride. The resulting derivative gives a four-fold increase in sensitivity compared to the published methods where trifluoroacetic anhydride was used for derivatization.     

Determination of 1,2-benzisoxazole-3-acetamidoxime hydrochloride (PE-257) in plasma using electron-capture gas chromatography

A gas chromatographic method has been developed that permits the accurate and specific determination of a new psychotropic agent, PF-257, in plasma. PF-257 is extracted with ethyl acetate from alkaline plasma and, after a clean-up procedure, derivatized with heptafluorobutyric anhydride to form 3-[(5-n-heptafluoropropyl-1,2, 4-oxadiazol-3-yl)methyl]-1,2-benzisoxazole (HOMB). The HOMB is assayed on a gas chromatograph equipped with an electron-capture detector. Accurate determinations of PF-257 are possible in the concentration range from 1-40 ng/ml with a relative standard deviation of 6.8%. The minimum detectable concentration in plasma is 0.1 ng/ml. Plasma levels of PF-257 in rats receiving intravenous or oral dosing (10 mg/kg) were determined.     

Stereospecific high-performance liquid chromatographic determination of tocainide

A sensitive high-performance liquid chromatographic assay was developed for the determination of tocainide enantiomers in plasma. Following extraction of tocainide from plasma, the enantiomers were derivatized with S-(+)-1-(1-naphthyl)ethylisocyanate. The resulting diastereomers were separated and quantified using normal-phase chromatography with fluorescence detection set at 220/345 nm (excitation/emission). The peaks, resolved with a resolution factor greater than 1.5, were free from interference. Linearity was established over the concentration range 0.25-10.0 mg/l for each enantiomer in plasma (r2 greater than 0.998). The inter-assay variability was less than 10% at all concentrations examined. The method can be used to determine the pharmacokinetics of tocainide enantiomers in man.     

Gas Chromatographic Analysis of Cyclophosphamide in Plasma and Tissues Using Nitrogen-Phosphorus Detection

Abstract

A gas chromatographic method for the analysis of cyclophosphamide in plasma, blood, and organ tissues is described. This method involves extraction of aliquots of plasma or tissue homo-genate in alkaline condition with ether. The extracted drug is derivatized with heptafluorobutyric anhydride followed by gas chromatographic separation via a glass column of 183 cm × 2 mm i. d. packed with 3% SE-30 on chromosorb W-HP. The derivatized cyclophosphamide and isophosphamide, an added internal standard, are detected by a nitrogen-phosphorus detector. The sensitivity limit of this method is 10 ng per gm or ml of sample and gives linearity over 100-fold of concentration range.     

Analytical and semi-preparative high-performance liquid chromatographic separation and assay of hydroxychloroquine enantiomers.

(+/-)-Hydroxychloroquine (HCQ) is an antimalarial and anti-arthritic drug which is administered as the racemate. An accurate, precise and sensitive high-performance liquid chromatographic assay was developed for the determination of HCQ enantiomers in samples from human plasma, serum, whole blood, and urine. After addition of (+/-)-chloroquine (internal standard), samples of blood component (0.5 ml) or urine (0.1 ml) were alkalinized and extracted with 5 ml of diethyl ether. After solvent evaporation the residues were derivatized with (+)-di-O-acetyl-L-tartaric anhydride at 45 degrees C for 30 min. The resulting diastereomers were then resolved using a C8 analytical column with a mobile phase consisting of 0.05 M KH2PO4 (pH 3)-methanol-ethanol-triethylamine (78:22:1:0.08). The ultraviolet detection wavelength was set at 343 nm. The derivatized HCQ enantiomers eluted in less than 40 min, free of interfering peaks. Excellent linear relationships (r2 > 0.997) were obtained between the area ratios and the corresponding plasma concentrations over a range of 12.5-500 ng/ml. The diastereomers could be hydrolysed using microwave energy and neutral pH, which enabled us to resolve the enantiomers on a semi-preparative (C18 column) scale. The method was suitable for the analysis and semi-preparative separation of HCQ enantiomers.     

Determination of fluoxetine enantiomers in rat plasma by pre-column fluorescence derivatization and column-switching high-performance liquid chromatography

A column-switching HPLC method employing both octadecylsilica (ODS) and chiral columns with fluorescence detection was developed for the determination of enantiomer of fluoxetine (FLX), an antidepressant drug, in rat plasma. Racemic FLX was derivatized with a fluorescent reagent, 4-(N-chloroformylmethyl-N-methyl)amino-7-nitro-2,1,3-benzoxadiazole (NBD-COCl) or 4-(N-chloroformylmethyl-N-methyl)amino-7-N,N-dimethylaminosulfonyl-2,1,3-benzoxadiazole (DBD-COCl) and the enantiomeric separation of the resultant derivatives was examined on an amylose-based chiral column (CHIRALPAK AD-RH) in the reversed-phase mode. The derivative with NBD-COCl (NBD-FLX) showed a sufficient separation factor (a) and resolution (Rs) compared with that with DBD-COCl. Thus, FLX was derivatized with NBD-COCl and the resultant NBD-FLX was first quantified on the ODS column and then introduced to the CHIRALPAK AD-RH column via a six-port switching valve to examine the enantiomeric ratio. The intra- and inter-day accuracy (97.6-112.7%) and precision (1.47-10.60%) were satisfactory in the range 10-1000 nM FLX and the limit of quantification was approximately 10 nM. The absolute recoveries of FLX with hexane from rat plasma were in the range 87.5-92.2% (n = 3). The method was applied to determine FLX enantiomers in the plasma of rats administered FLX orally, and it was shown that the R-isomer was eliminated faster than the S-isomer.     

Quantitative determination of SC-68328 in dog plasma using flow injection and tandem mass spectrometry

A flow injection/tandem mass spectrometric assay was developed to quantitate SC-68328 in dog plasma using its stable isotopic analog [13C4]SC-68328 as an internal standard (IS). Since SC-68328, a manganese-based superoxide dismutase mimetic, is very unstable, very polar and adheres to silica-based high-performance liquid chromatographic columns, the analyte and IS were derivatized to their bis-isothiocyanate forms followed by a liquid-liquid extraction with methylene chloride and analyzed using positive ion electrospray mass spectrometric detection. SC-68328 was quantitated using the peak-height ratio of SC-68328 to its IS using MS/MS in the multiple reaction monitoring mode. The lower limit of quantitation of the assay was 0.25 microg ml(-1) SC-68328 in dog plasma with an inter-day precision of 11.8% and an accuracy of 113% (n = 12). Acceptable precision and accuracy were also obtained for concentrations in the calibration curve range (0.25-10 microg ml(-1) SC-68328 in dog plasma).     

Rapid determination of lysine in biological samples by isocratic liquid chromatography.

A simple, rapid, and sensitive method was developed for detection and quantitation of lysine (Lys) in various biological samples by isocratic liquid chromatography (LC). Samples containing Lys and other amino acids were derivatized with 9-fluorenylmethyl chloroformate (FMOC-CI). The mobile phase used for isocratic elution was 50 mmol/L sodium acetate buffer (pH 4.20)-acetonitrile (43 + 57, v/v). Lys was detected with a UV detector at 265 nm. The derivatized Lys eluted from a LiChrospher 100 RP-18 (150 x 4.0 mm id) column at a retention time of 5.6 min. The limit of detection was 0.73 mumol/L (signal-to-noise [S/N] ratio, 3:1), and the limit of quantitation was 2.37 mumol/L (S/N ratio, 10:1). Lys recoveries from fortified biological samples were > 97.5%. Average Lys contents found in rumen fluid samples collected before the morning feeding and at 2.0, 4.0, and 6.0 h after feeding were 4.26, 3.34, 3.58, and 3.82 mumol/L, respectively. The hydrolysate of a sample of mixed rumen microorganisms collected before the morning feeding was determined to contain 1.372 mumol/mg microbial nitrogen in the form of Lys. The Lys concentrations of human plasma, goat plasma, human urine, and goat urine were 140.0, 102.0, 58.0, and 32.0 mumol/L, respectively.     

RP-HPLC assay for 1,2-5,6-dianhydro-3,4-disuccinyl-galactitol and its metabolites in blood plasma and liver

Summary A method involving pre-column derivatization and HPLC assay is described for measuring submicrogram quantities of 1,2-5,6-dianhydro-3,4-disuccinyl-galactitol (1,2-5,6-dianhydro-3,4-bis(carboxypropionyl)-galactitol), an effective cytostatic drug and its metabolites in blood plasma and liver homogenate. The substance and its metabolites were derivatized with sodium pentamethylene-dithiocarbamate to form different bis(dithiocarbamoyl) esters, which can be detected by UV absorbance at 254 and 280 nm. The directly derivatized products were then extracted into CHCl₃, and after sample preparation resolved by RP-HPLC on SAS-Hypersil column.     

气相色谱-质谱法测定血浆中双氯芬酸钠的含量

提出了应用气相色谱-质谱法测定血浆中双氯芬酸钠含量。血浆样品中双氯芬酸钠经盐酸提取,并加入三甲基硅烷衍生化试剂进行衍生化,所得衍生化产物经萃取、净化后供气相色谱分离及质谱测定。采用选择离子质谱扫描方式,供试品双氯芬酸钠-三甲基硅烷衍生物定量选择离子检测质荷比为m/z367,内标物布洛芬-三甲基硅烷衍生物定量选择离子检测质荷比为m/z263。双氯芬酸钠的线性范围为0.005～5.0mg·mL-1,检出限(3S/N)为0.5μg.L-1。方法回收率在94.6%～97.4%之间,日内、日间相对标准偏差(n=6)均小于5%。     

Determination of xylometazoline in plasma and urine by gas chromatography using a fused-silica capillary column and an electron-capture detector

A sensitive method is described for the determination of unchanged xylometazoline in plasma and urine at concentrations down to 35 nmol/l. After addition of naphazoline as an internal standard, both compounds are extracted with dichloromethane-diethyl ether (20:80) at pH 10, back-extracted with an acidic solution and re-extracted from a sodium hydroxide solution with dichloromethane-diethyl ether (20:80). The compounds are then derivatized with heptafluorobutyric anhydride in the presence of pyridine. The derivatives are determined by capillary gas chromatography using electron-capture detection.     

Direct enantiomeric analysis of amphetamine in plasma by simultaneous solid phase extraction and chiral derivatization

Summary An analytical approach has been developed for the one step determination of enantiomeric amphetamine composition in plasma, using on-line, pre-column solid phase derivatization with reversed phase HPLC separation. The high molecular weight protein components were excluded by the small pore structure of the polymer and washed out of the reaction column before derivatization. Spiked amphetamine in human plasma was extracted and derivatized by the polystyrene based FMOC-L-prolyl solid phase reagent. The derivatized diastereomers were separated on a conventional ODS column with an ACN/H₂O mobile phase. No kinetic resolution or racemization was observed in this solid phase derivatization. Calibration plots and reproducibility experiments were performed to demonstrate the validity of the new approach. Automation of the procedure provided a simple and reproducible method for direct chiral recognition in plasma samples.     

Determination of a new blood glucose-lowering agent in biological fluids by capillary gas chromatography using electron-capture detection

A gas chromatographic method for the determination of 5-(4-chlorophenyl)-2-[(4-methylphenyl)sulphonyl]-4-pentynoic acid (I) in plasma (serum) and urine has been developed. After an extraction process, the cleaned-up organic extract was derivatized with diazomethane at ambient temperature. Results are evaluated from peak-height ratios with respect to the appropriate internal standard. The detection limit following extraction of a 1-ml plasma sample is about 20 ng/ml.     

Determination of N-acetylcysteine in human plasma by gas chromatography-mass spectrometry

An automatic mass spectrometric method for the quantitation of N-acetylcysteine (NAC) in human plasma has been developed. NAC was extracted from plasma with ethyl acetate and derivatized in two steps with 2-propanol and pentafluoropropionic anhydride. The volatile derivative obtained was ideal for gas chromatographic-mass spectrometric analysis. Data obtained by analysing the plasma of healthy volunteers to whom 600 mg of NAC had been orally given are reported.     

9-Fluoreneacetyl-tagged, solid-phase reagent for derivatization in direct plasma injection.

We describe here a resin-based derivatization reagent, containing a 9-fluoreneacetyl tag on a controlled-pore substrate, for direct injection analysis of amphetamine in plasma. On-line, pre-column derivatization was performed by direction injection of diluted plasma sample into an sodium dodecyl sulfate-containing mobile phase. Amphetamine was trapped in the hydrophobic derivatization column and derivatized at elevated temperature by the activated solid-phase reagent. The derivatized 9-fluoreneacetyl amphetamide was separated by reversed-phase high-performance liquid chromatography with a step gradient and determined by fluorescence detection. The synthesis scheme, characterization, and optimization of the derivatization conditions for the solid-phase reagent are described. The method was evaluated by reproducibility tests and single blind spiking analysis. This solid-phase reagent combined with a surfactant containing mobile phase provided a sensitive and simple procedure for on-line derivatization in direct injection analysis of biological fluids.     

Quantitative analysis of memantine in human plasma by gas chromatography/negative ion chemical ionization/mass spectrometry

A sensitive and specific method for the determination of memantine in human plasma is presented. Memantine was extracted from plasma and derivatized to the pentafluorobenzoyl derivative in a one-step procedure avoiding any sample concentration steps. Amantadine was used as an internal standard. The compounds were measured by gas chromatography/negative ion chemical ionization mass spectrometry without any further processing. Using this detection mode, the fragment ions at m/z 353 and 325 were obtained at high relative abundance. Calibration graphs were linear over the range 0.117-30 ng ml(-1). At the limit of quantification (LOQ), the inter-assay precision was 2.00% and the intra-assay variability was 3.22%. The accuracy at the LOQ showed deviations of -1.42% (intra-assay) and -2.47% (inter-assay). The method is rugged, rapid and robust and was applied to the batch determination of memantine during pharmacokinetic profiling of the drug.