Sensitive gas-liquid chromatographic method for the assay of the neuroleptic drug cis(Z)-flupentixol in human serum or plasma

A gas-liquid chromatographic (GLC) assay suitable for the analysis of the cis(Z)-stereoisomer of the antipsychotic drug flupentixol in human serum or plasma was developed. The minimal quantifiable concentration was 0.5 ng/ml and the day-to-day coefficient of variation was 11.2% at 1 ng/ml and 8.7% at 10 ng/ml. Following addition of perphenazine as the internal standard (I.S.) and aqueous NaOH, samples (2 ml) are extracted with n-hexane-isoamyl alcohol (98.5:1.5, v/v) (solvent), back-extracted to 0.1 M HCl and after one washing-step and addition of aqueous NaOH again extracted into 100 μl solvent. After evaporation to dryness, the extract is reconstituted in 20 μl solvent and evaporated to approximative 10 μl. A 4-μl aliquot is injected cool on-column onto the GLC system. A gas chromatograph HP 5890 with on-column injection port, nitrogen-phosphorus detector (NPD), a HP-1 25 m × 0.32 mm I.D., 0.5 μm capillary and hydrogen (3 ml/min, automated pressure control) as the carrier gas was applied. The negative influence of light on the assay was measured and discussed. The suitability of this method for clinical pharmacokinetic studies and therapeutic drug monitoring (TDM) was determined by the analysis of serum samples of 12 schizophrenic patients.     

Determination of MDMA and MDA in rat urine by semi-micro column HPLC-fluorescence detection with DBD-F and their monitoring after MDMA administration to rat.

A simultaneous semi-micro column HPLC method with fluorescence detection of abused drugs, such as 3,4-methylenedioxymethamphetamine (MDMA), 3,4-methylenedioxyamphetamine (MDA), amphetamine (AP) and methamphetamine (MP) in rat urine was examined by using 4-(N,N-dimethylaminosulphonyl)-7-fluoro-1,2,3-benzoxadiazole (DBD-F) as a labelling reagent and alpha-phenylethylamine as an internal standard (IS). A sample (50 microL) of rat urine was added to 5 microL IS and 100 microL 100 mmol/L borate buffer (pH 12) and extracted with 1.5 mL n-hexane. After evaporation, 50 microL 75 mmol/L borate buffer (pH 8.5) and 50 microL 20 mmol/L DBD-F in CH3CN were added to the residue and mixed well. The resultant solution was heated for 20 min at 80 degrees C and then cooled in an ice bath. A good separation of DBD-derivatives could be achieved within 45 min using a semi-micro ODS column with an eluent of CH3CN/CH3OH/10 mmol/L imidazole-HNO3 buffer (pH 7.0) (= 45:5:50, v/v/v %). The DBD derivatives were monitored at 565 nm with an excitation at 470 nm. The calibration curves showed good linearity (r = 0.997) with 0.5-15 ng/mL detection limits at a S/N ratio of 3. MDMA and MDA in rat urine could be monitored for 15 h after a single administration of MDMA to rat (2.0 mg/kg, i.p.). The concentrations for MDMA and MDA (n = 3) were 0.13-160.1 and 0.17-10.9 microg/mL, respectively.     

Improved gas chromatographic procedure for the determination of clonazepam levels in plasma using a nitrogen-sensitive detector

A gas—liquid chromatographic procedure (GLC) is described for the determination of clonazepam in plasma. The drug is extracted from buffered plasma at pH 9.0 with diethyl ether and then back-extracted into 6 N hydrochloric acid—6 N sulfuric acid (95:5) and hydrolyzed at 100°C to convert the drug into its benzophenone derivative. The benzophenone derivative of flurazepam is added to plasma as an internal reference standard. Drug derivatives are finally extracted from the neutralized aqueous phase and assayed by GLC. The present procedure makes use of a nitrogen-sensitive detector which is more stable and selective than the commonly employed electron-capture procedure. The sensitivity of the detector for clonazepam is 1 ng/ml.     

Methyl malondialdehyde is not suitable as an internal standard for malondialdehyde detection in urine after derivatisation with 2,4-dinitrophenylhydrazine

A previously described method of measurement of malondialdehyde (MDA) in human urine after derivatisation with 2,4-dinitrophenylhydrazine (DNPH) was tested for a possibility of using methyl malondialdehyde (MeMDA) as an internal standard. Despite structural similarity, those compounds were found to produce different yields of derivatisation under the same conditions depending on urine matrix. We conclude, that MeMDA is not suitable as an internal standard for the measurement of MDA in urine under previously reported conditions when DNPH is used as a deriviatising agent.     

Mutagenicity of the malondialdehyde oligomerization products 2-(3'-oxo-1'-propenyl)-malondialdehyde and 2,4-dihydroxymethylene-3-(2,2-dimethoxyethyl)glutaraldehyde in Salmonella

Malondialdehyde (MDA), a byproduct of non-enzymatic lipid peroxidation and prostaglandin biosynthesis, has been shown to be a weak frameshift mutagen in Salmonella mutagenicity assays. Because it is a dialdehyde, MDA can undergo self condensation to form polymeric products. It is possible that these condensation products are highly mutagenic and have contributed to previously reported estimates of MDA mutagenicity. We synthesized two major MDA polymerization products, (1) 2-(3'-oxo-1'-propenyl)-malondialdehyde [(MDA)2] and (2) 2,4-dihydroxymethylene-3-(2,2-dimethoxyethyl)glutaraldehyde [(MDA)3Me2] and tested their mutagenicity in the Salmonella frameshift tester strains hisD3052 and TA94 (hisD3052/pKM101). Analysis of the reversion rates revealed both (MDA)2 and (MDA)3Me2 to be weak mutagens, approximately equipotent to MDA. Although both (MDA)2 and (MDA)3Me2 are mutagenic, the fact that their formation is thermodynamically unfavorable under physiological conditions suggests they do not contribute significantly to the mutagenicity of MDA solutions.     

Preparative steps necessary for the accurate measurement of malondialdehyde by high-performance liquid chromatography.

The need for a more specific, reliable, and reproducible technique for the measurement of malondialdehyde (MDA) has prompted modifications of currently available methods based on the formation and recovery of the complex between MDA and thiobarbituric acid (TBA). To 500 microliters of plasma or to 300 mg of liver homogenate, 2 ml of H2O and 500 microliters of 0.5% butylated hydroxytoluene in methanol were added to prevent further formation of MDA. Precipitation of proteins carried out with 200 microliters of 0.66 N H2SO4 and 150 microliters of 10% Na2WO4 (w/v) led to complete recovery of the MDA standard. Maximum formation of the MDA-TBA complex was obtained by adjusting the pH between 2.5 and 4.5 and heating the MDA-TBA mixture at 100 degrees C for 60 min. Extraction of the MDA-TBA complex was a critical step and proved complete with n-butanol at pH less than 0.75. It was then evaporated at 37 degrees C under nitrogen. The MDA-TBA complex solubilized in H2O was shown to be stable for at least 7 days. These preparative steps led to the detection of a single peak that on spectral analysis was identified as pure MDA-TBA. This procedure offers several advantages in terms of specificity, recovery, and reproducibility.     

Simple quantification of lansoprazole and rabeprazole concentrations in human serum by liquid chromatography/tandem mass spectrometry

A rapid, simple and highly sensitive method was developed for the quantitative determination of lansoprazole and rabeprazole concentrations in 20 microL of human serum using high-performance liquid chromatography/tandem mass spectrometry (LC/MS/MS). Analytes, along with an internal standard (lansoprazole deuterium derivatives), were separated using a mobile phase of acetonitrile/1mM ammonium formate (140/60, v/v) on a C18 analytical column and analyzed in the selected reaction-monitoring (SRM) mode. The lower limit of quantification was 0.25 ng/mL. A good linear response was observed for each analyte (from 0.25 ng to 2.5 microg/mL). This method was useful for therapeutic drug monitoring and pharmacokinetic studies.     

Improved method for plasma malondialdehyde measurement by high-performance liquid chromatography using methyl malondialdehyde as an internal standard

Measurement of malondialdehyde (MDA) is an important contribution to the assessment of oxidative stress. We report a sensitive and reproducible high-performance liquid chromatography (HPLC) method for measurement of plasma MDA in the assessment of lipid peroxidation. Using methyl malondialdehyde (Me-MDA) as an internal standard with reversed-phase HPLC and UV detection and derivatisation with 2,4 dinitrophenylhydrazine (DNPH), we obtained maximum MDA values with 60-min incubation of 10% plasma with 1 M NaOH at 60 degrees C. The dilution of the plasma and a longer incubation time in the alkaline hydrolysis step greatly improved recovery of MDA from its bound form. Ratios of peak height of MDA/Me-MDA were linear over a range of 0-100 microM with correlation coefficients >0.99. The recovery was 88.5%. Within and between run variations were <4 and <7%, respectively. The mean MDA value measured in 20 healthy volunteers was 13.8 microM (+/-1.32).     

Quantitation of lysophosphatidylcholine molecular species in rat cardiac tissue.

We have developed a rapid and sensitive procedure for isolation and measurement of 1-acyllysophosphatidylcholine (LPC) species in rat myocardial tissue. Tissues were spiked with heptadecanoyl-LPC internal standard and extracted with chloroform/methanol. The chloroform phase was dried, resuspended in chloroform/propan-2-ol (2/1, v/v), and applied to an aminopropyl-bonded phase (Bond Elut) column. Following stepwise elution with several solvent mixtures, the LPC fraction (ethyl acetate/methanol, 4/6, v/v) was separated by HPLC with direct quantitation of palmitoyl-LPC (P-LPC), oleoyl-LPC (O-LPC), and stearoyl-LPC (S-LPC), using an evaporative light scattering mass detector. Calibration curves were generated for each individual LPC species. Recoveries of added [14C]LPC and of heptadecanoyl-LPC internal standard after extraction and chromatography were 85.8 +/- 1.9% (mean +/- SE, N = 10) and 83.4 +/- 1.8% (N = 15), respectively. This assay showed satisfactory sensitivity, reproducibility, and accuracy for measurement of LPC species in rat myocardial tissue. The major molecular species of LPC in rat myocardium were found to be P-LPC and S-LPC, which were two- to sixfold as abundant as O-LPC. In isolated, crystalloid-perfused rat hearts the time of perfusion was found to significantly influence the content of P-LPC (0 min, 252 +/- 10; 15 min, 178 +/- 10, P less than 0.001, compared with 0 min; 40 min, 131 +/- 4, P less than 0.001; and 70 min, 129 +/- 4, P less than 0.001; nmol/g dry weight), but not the content of O-LPC and S-LPC. The method will be useful for studying the participation of LPC species in physiology, pathophysiology, and therapeutics.     

Liquid chromatography-electrospray tandem mass spectrometric assay suitable for quantitation of halofuginone in plasma

An LC-MS/MS method was developed to quantitate the potential antitumor agent halofuginone in plasma. The assay uses 0.2 ml of plasma; chlorohalofuginone internal standard; acetonitrile for protein precipitation; a Phenomenex SYNERGI 4 micro Polar RP 80A (4 microm, 100 mm x 2 mm) column; an isocratic mobile phase of methanol:water:formic acid (80:20:0.02, v/v/v); and positive-ion electrospray ionization with selective reaction monitoring detection. Halofuginone eluted at approximately 2.4 min, internal standard eluted at approximately 2.9 min, and no endogenous materials interfered with their measurement. The assay was accurate, precise, and linear between 0.1 and 100 ng/ml. Halofuginone could be quantitated in dog plasma for at least 24 h after an i.v. dose of 0.1mg/kg. The assay is being used in ongoing pharmacokinetic studies of halofuginone.     

Liquid chromatography-electrospray mass spectrometry determination of free and total concentrations of ropivacaine in human plasma

A specific and sensitive liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) method was developed for the determination of free and total ropivacaine in human plasma. The work-up procedure involved a simple precipitation of plasma proteins with methanol. Etidocaine served as the internal standard. After microscale equilibrium-dialysis, measurement of free ropivacaine levels was performed after direct injection of the dialysate into the chromatograph. The system used a Zorbax eclipse XD8 C8 analytical column packed with 5 microm diameter particles as the stationary phase. The mobile phase consisted of a 15-min gradient (mobile phase A: 0.05% (v/v) trimethylamine in acetonitrile, mobile phase B: 2mM ammonium formate buffer (pH 3)). Mass spectrometric data were acquired in single ion monitoring mode at m/z 275 for ropivacaine and m/z 277 for etidocaine. The drug/internal standard peak area ratios (plasma) or peak areas (dialysate) were linked via a quadratic relationship to concentrations. Precision ranged from 1 to 7.6% accuracy was between 92.6 and 109%. The lower limits of quantitation were 1 microg/l in plasma and 2 microg/l in the dialysate. This method was found suitable for the analysis of plasma samples collected during a clinical trial performed in 30 infants undergoing epidural anaesthesia or continuous psoas compartment block.     

Quantitative measurement of propofol and in main glucuroconjugate metabolites in human plasma using solid phase extraction-liquid chromatography-tandem mass spectrometry

A high-performance liquid chromatographic method coupled with tandem mass spectrometry detection has been developed for the determination of propofol and its main glucuroconjugate metabolites (propofol-glucuronide (PG), 1-quinol-glucuronide (1-QG) and 4-quinol-glucuronide (4-QG) in human plasma. All compounds were extracted with a single solid phase extraction procedure using Max Oasis cartridges. Propofol and thymol (internal standard) were analyzed using a C8 reversed-phase column with a mobile phase consisting of methanol-water (75:25, v/v) containing 0.025% NH(4)OH. Chromatography of glucuroconjugate metabolites and phenyl-beta-d-glucuronide (internal standard) was performed using a hydrophilic interaction liquid chromatography (HILIC) and a mixture of acetonitrile/water/ammonium acetate buffer (100 mM, pH 5, 87/1/12, v/v/v). Both chromatographic separations were achieved in isocratic mode allowing a rapid analysis without re-equilibration of the phase. The method is specific and sensitive with a range of 10-1500 ng mL(-1) for propofol and 1-QG, 20-3000 ng mL(-1) for PG and 25-3750 ng mL(-1) for 4-QG. The regression curves were linear for all compounds. The method is accurate and precise with intra-assay and inter-assay precision <8% and bias < or =6% for all compounds. This assay has allowed the successful measurement of propofol and its main glucuroconjugate metabolites in human plasma from 24 patients undergoing anaesthesia for elective partial hepatectomy surgery.     

The analysis of acyl-coenzyme A derivatives by reverse-phase high-performance liquid chromatography

A method for determining tissue levels of Coenzyme A and various short-chain-length acyl-CoA derivatives using high-performance liquid chromatography is presented. Separation of the various compounds was accomplished using a reverse-phase Spherisorb ODS II, 5-microns C18 column. Mobile-phase solvents were (a) potassium phosphate, 220 mM; thiodiglycol (2,2-thiodiethanol), 0.05% (v/v), pH 4.0 and (b) methanol, 98%; chloroform; 2% (v/v). The various acyl-CoA derivatives were detected by monitoring the column effluent at 254 nm. Nearly baseline separation was obtained for a standard mixture of free CoASH, methylmalonyl-CoA, beta-hydroxy-beta-methylglutaryl-CoA, succinyl-CoA, acetoacetyl-CoA, acetyl-CoA, propionyl-CoA, isobutyryl-CoA, beta-methyl-crotonyl-CoA, and isovaleryl-CoA. CoA derivative profiles were determined in neutralized perchloric acid extracts of perfused rat hearts and livers and of isolated rat liver mitochondria to demonstrate the utility of this method for assessing the levels of CoA derivatives in biological samples.     

Determination of taurine in plasma by high-performance liquid chromatography using 4-(5,6-dimethoxy-2-phthalimidinyl)-2-methoxyphenylsulfonyl chloride as a fluorescent labeling reagent

A sensitive high-performance liquid chromatography method for the determination of taurine in human plasma was developed. Taurine and N-methyltaurine (internal standard) were derivatized with 4-(5,6-dimethoxy-2-phthalimidinyl)-2-methoxyphenylsulfonyl chloride to produce fluorescent sulfonamides. The labeling reaction was carried out at 70 degrees C for 20 min at pH 7.5. The fluorescent derivatives were separated on a reversed-phase column by a stepwise elution using (A) acidic phosphate buffer/acetonitrile (83/17) and (B) acetonitrile and detected by fluorescence measurement at excitation and emission wavelengths of 318 and 392 nm, respectively. The detection limit (signal-to-noise ratio=3) of taurine was 3 fmol per injection. The within-day and day-to-day relative standard deviations were 3.0-4.8 and 2.5-4.7%, respectively. The concentration (means) of taurine in normal human plasma was 48.9+/-7.5 microM.     

Quantitation of platelet-activating factor by high-performance liquid chromatography with fluorescent detection

Platelet-activating factors, 1-O-hexadecyl- and 1-O-octadecyl-2-acetyl-sn-glycero-3-phosphocholine (C16-AGEPC and C18AGEPC), were measured by reverse-phase high-performance liquid chromatography with fluorescent detection. C16AGEPC, C18AGEPC, and 1-O-hexadecyl-2-propionyl-sn-glycero-3-phosphocholine, which was suitable for use as an internal standard, were hydrolyzed with phospholipase C, and then the resulting hydrolyzed products were derivatized with 7-methoxycoumarin-3-carbonyl chloride or 7-methoxy-coumarin-4-acetic acid to form 7-methoxycoumarin ester derivatives which permit a fluorometric detection. The lower limit of detection of the derivatives was about 100 pg at a signal-to-noise ratio of 5:1. A commercial platelet-activating factor was demonstrated to contain C16AGEPC (70%) and C18AGEPC (12.8%) by the present method. The present method was also applicable to the measurement of acetyl-CoA:1-alkyl-2-lyso-sn-glycero-3-phosphocholine acetyltransferase activity in a lysate of human polymorphonuclear leukocytes.     

Liquid chromatography-electrospray mass spectrometry determination of ibogaine and noribogaine in human plasma and whole blood. Application to a poisoning involving Tabernanthe iboga root

A liquid chromatography/electrospray ionization mass spectrometry (LC-ESI-MS) method was developed for the first time for the determination of ibogaine and noribogaine in human plasma and whole blood. The method involved solid phase extraction of the compounds and the internal standard (fluorescein) from the two matrices using OasisHLB columns. LC separation was performed on a Zorbax eclipse XD8 C8 column (5 microm) with a mobile phase of acetonitrile containing 0.02% (v/v) trimethylamine and 2mM ammonium formate buffer. MS data were acquired in single ion monitoring mode at m/z 311.2, 297.2 and 332.5 for ibogaine, noribogaine and fluorescein, respectively. The drug/internal standard peak area ratios were linked via a quadratic relationship to plasma (0.89-179 microg/l for ibogaine; 1-200 microg/l for noribogaine) and to whole blood concentrations (1.78-358 microg/kg for ibogaine; 2-400 microg/kg for noribogaine). Precision ranged from 4.5 to 13% and accuracy was 89-102%. Dilution of the samples had no influence on the performance of the method. Extraction recoveries were > or =94% in plasma and > or =57% in whole blood. The lower limits of quantitation were 0.89 microg/l for ibogaine and 1 microg/l for noribogaine in plasma, and 1.78 microg/kg for ibogaine and 2 microg/kg for noribogaine in whole blood. In frozen plasma samples, the two drugs were stable for at least 1 year. In blood, ibogaine and noribogaine were stable for 4h at 4 degrees C and 20 degrees C and 2 months at -20 degrees C. The method was successfully used for the analysis of a poisoning involving Tabernanthe iboga root.     

Liquid chromatographic-tandem mass spectrometric method for the quantitation of huperzine A in dog plasma

A rapid and sensitive LC-MS-MS method for the determination of huperzine A in dog plasma using huperzine B as internal standard has been developed and validated. The analyte and internal standard were extracted from plasma using n-hexane-dichloromethane-2-propanol (300:150:15, v/v/v), chromatographed on a C(18) column (5 microm, 50 mm x 4.6 mm i.d.) with a mobile phase consisting of acetonitrile-methanol-10mM ammonium acetate (35:40:25, v/v/v), and detected using a tandem mass spectrometer with a TurboIonSpray ionization interface. The run time was only 2 min. The assay was linear over the concentration range 0.05-20 ng/ml and intra- and inter-day precision over this range were <5.3% with good accuracy. The limit of detection in plasma was 0.01 ng/ml. The method was successfully applied to define plasma concentration-time curves of huperzine A in dogs after the last dose of an intramuscular injection (10 microg/kg per day for 15 days) of a sustained-release formulation of huperzine A.     

Determination of urinary malondialdehyde by isotope dilution LC-MS/MS with automated solid-phase extraction: a cautionary note on derivatization optimization

A highly sensitive quantitative LC-MS/MS method was developed for measuring urinary malondialdehyde (MDA). With the use of an isotope internal standard and online solid-phase extraction, urine samples can be directly analyzed within 10 min after 2,4-dinitrophenylhydrazine (DNPH) derivatization. The detection limit was estimated as 0.08 pmol. This method was further applied to assess the optimal addition of DNPH for derivatization and to measure urinary MDA in 80 coke oven emission (COE)-exposed and 67 nonexposed workers. Derivatization optimization revealed that to achieve complete derivatization reaction, an excess of DNPH is required (DNPH/MDA molar ratio: 893-8929) for urine samples that is about 100 times higher than that of MDA standard solutions (molar ratio: 10-80). Meanwhile, the mean urinary concentrations of MDA in COE-exposed workers were significantly higher than those in nonexposed workers (0.23±0.17 vs 0.14±0.05 μmol/mmol creatinine, P<0.005). Urinary MDA concentrations were also significantly associated with the COE (P<0.005) and smoking exposure (P<0.05). Taken together, this method is capable of routine high-throughput analysis and accurate quantification of MDA and would be useful for assessing the whole-body burden of oxidative stress. Our findings, however, raise the issue that derivatization optimization should be performed before it is put into routine biological analysis.     

Determination of 7-ketocholesterol in plasma by LC-MS for rapid diagnosis of acid SMase-deficient Niemann-Pick disease

Acid sphingomyelinase (ASMase)-deficient Niemann-Pick disease (NPD) is caused by mutations in the sphingomyelin phosphodiesterase 1 (SMPD1) gene, resulting in accumulation of sphingomyelin in the lysosomes and secondary changes in cholesterol metabolism. We hypothesized that the oxidation product of cholesterol, 7-ketocholesterol (7-KC), might increase in the plasma of patients with ASMase-deficient NPD. In this study, a rapid and nonderivatized method of measurement of plasma 7-KC by liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed. Plasma samples from healthy subjects, patients with ASMase-deficient NPD, nonaffected ASMase-deficient NPD heterozygotes, Niemann-Pick type C (NPC) disease, glycogen storage disorder type II (GSDII), Gaucher disease (GD), mucopolysaccharidosis type II (MPSII), Krabbe disease (KD), and metachromatic leukodystrophy (MLD) were tested retrospectively. Markedly elevated 7-KC was found in patients with ASMase-deficient NPD and NPC disease that showed significant differences from ASMase-deficient NPD heterozygotes; patients with GSDII, GD, MPSII, KD, and MLD; and normal controls. The analysis of plasma 7-KC by LC-MS/MS offers the first simple, quantitative, and highly sensitive method for detection of ASMase-deficient NPD and could be useful in the diagnosis of both ASMase-deficient NPD and NPC disease.     

A simple and sensitive HPLC method for determination of gliclazide in human serum

A simple, rapid and specific method for analysis of gliclazide in serum by a sensitive high-performance liquid chromatographic method is described. Only 100 microl of serum and a little sample work-up is required. A simple procedure of extraction by toluene followed by evaporation to dryness under a gentle stream of air and dissolving the dried residue in mobile was used. The gliclazide peak was separated from endogenous peaks on a C(8) column by a mobile phase of acetonitrile-water (45:55, v/v), pH 3. Gliclazide and internal standard (phenytoin) were eluted at 6.8 and 3.8 min, respectively. The limit of quantitation (LOQ) for gliclazide in serum was 75 ng/ml at 230 nm. The method was linear over the range of 75-10,000 ng/ml with r(2) of 0.999. Mean recovery for gliclazide and internal standard was 84.5 and 87%, respectively.