Analysis of lipoic acid in biological samples by gas chromatography with flame photometric detection

A selective and sensitive gas chromatographic method for the analysis of lipoic acid in biological samples has been developed. After base hydrolysis of the sample, the liberated lipoic acid was converted into its S,S-diethoxycarbonyl methyl ester derivative and measured by gas chromatography using a DB-210 capillary column and a flame photometric detector. The calibration curve was linear in the range 20–500 ng, and the detection limit was ca. 50 pg injected. The best hydrolysis conditions for the biological samples were obtained by using 2 M potassium hydroxide containing 4% bovine serum albumin at 110°C for 3 h. Using this method, lipoic acid in the hydrolysate could be selectively determined without any interference from matrix substances. Analytical results for the determination of lipoic acid in the mouse tissue and bacterial cell samples are presented.     

Simultaneous determination of norethindrone and ethinyl estradiol in human plasma by high performance liquid chromatography with tandem mass spectrometry--experiences on developing a highly selective method using derivatization reagent for enhancing sensitivity

In the present work, for the first time, a liquid chromatographic method with tandem mass spectrometric detection (LC-MS/MS) for the simultaneous analysis of norethindrone, and ethinyl estradiol, was developed and validated over the concentration range of 50-10000pg/ml and 2.5-500pg/ml, respectively, using 0.5 ml of plasma sample. Norethindrone, ethinyl estradiol, and their internal standards norethindrone-(13)C2, and ethinyl estradiol-d4, were extracted from human plasma matrix with n-butyl chloride. After evaporation of the organic solvent, the extract was derivatized with dansyl chloride and the mixture was injected onto the LC-MS/MS system. The gradient chromatographic elution was achieved on a Genesis RP-18 (50 mm x 4.6 mm, 3 microm) column with mobile phase consisted of acetonitrile, water and formic acid. The flow rate was 1.0 ml/min and the total run time was 5.0 min. Important parameters such as sensitivity, linearity, matrix effect, reproducibility, stability, carry-over and recovery were investigated during the validation. The inter-day precision and accuracy of the quality control samples at low, medium and high concentration levels were <6.8% relative standard deviation (RSD) and 4.4% relative error (RE) for norethindrone, and 4.2% RSD and 5.9% RE for ethinyl estradiol, respectively. Chromatographic conditions were optimized to separate analytes of interest from the potential interference peaks, arising from the derivatization. This method could be used for pharmacokinetic and drug-drug interaction studies in human subjects.     

Quantitative analysis of polychlorinated biphenyls, organochlorine insecticides, polycyclic aromatic hydrocarbons, polychlorinated hydrocarbons and polynitrohydrocarbons in spiked samples of soil, water and plasma by selected-ion monitoring gas chromatography–mass spectrometry

A broad range of pollutants such as polycyclic aromatic hydrocarbons (PAHs), polychlorinated hydrocarbons (PCHs), polynitrohydrocarbons (PNHs), polychlorinated biphenyls (PCBs) and organochlorine (OCs) insecticides were simultaneously analyzed in spiked soil, water or plasma samples by using gas chromatography–mass spectrometry (GC–MS). Water and plasma samples containing the pollutants were extracted by a solid-phase extraction (SPE) method using florisil columns. The soil samples, fortified with the toxicants, were extracted with water, methanol or dichloromethane (DCM). The water extract was processed by the SPE method. The methanol and DCM samples were dried, dissolved in acetonitrile and subjected to the SPE extraction. The extracted samples were analyzed by GC–MS programmed to monitor selected ions. The deuterium labelled compounds were used as the internal standards. The chromatographic profile of total ions indicated complete separation of some compounds such as isophorone, naphthalene, all PCBs, most OC insecticides and PNHs; high M_r PAHs and some PCHs were partially or incompletely separated. The chromatographic profile of individual ion indicated good separation of each ion. The minimum detection limit ranged from 1 to 4 pg injected when 1 or 2 ions were monitored or from 20 to 200 pg injected when 20 ions were monitored. The SPE method that provided 60–105% recovery of pollutants from water samples, provided only 2–60% recovery from plasma samples. This may be due to the binding of pollutants to plasma proteins. Water recovered 1–30%, while methanol or DCM recovered 65–100% of the pollutants added to the soil samples. The use of internal standards corrected for the loss of pollutants from plasma or soil.     

Application of a sensitive liquid chromatographic/tandem mass spectrometric method to pharmacokinetic study of nalmefene in humans

A sensitive, specific and rapid liquid chromatographic/tandem mass spectrometric (LC/MS/MS) method was developed and validated for quantification of nalmefene in human plasma. An aliquot of 200 microL plasma sample was simply precipitated by 400 microL methanol. Separation of nalmefene and the internal standard hydromorphone from the interferences was achieved on a C(18) column followed by MS/MS detection. The analytes were monitored in the positive ionization mode with a TurboIonspray source. The method had a total chromatographic run time of 4.5 min and linear calibration curves over the concentration range of 10-5000 pg/mL. The lower limit of quantification (LLOQ) was 10 pg/mL. The intra- and inter-day precision was less than 10.1% determined from QC samples at concentrations of 30, 300 and 4500 pg/mL, and the accuracy was within +/-3.4%. As the method was more sensitive (10 times higher) than those reported previously, we investigated the pharmacokinetics of nalmefene in healthy volunteers after a single intravenous injection of low dose (30 microg) of nalmefene hydrochloride for the first time.     

Development and validation of a sensitive gas chromatography–ammonia chemical ionization mass spectrometry method for the determination of tabun enantiomers in hemolysed blood and plasma of different species

The aim of this study was to develop and validate a fast, sensitive and easily applicable GC–MS assay for the chiral quantification of the highly toxic organophosphorus compound tabun (O-ethyl-N,N-dimethylphosphoramidocyanidate, GA) in hemolysed swine blood for further use in toxicokinetic and toxicodynamic studies. These requirements were fulfilled best by a GC–MS assay with positive chemical ionization with ammonia (GC–PCI-MS). Separation was carried out on a β-cyclodextrin capillary column (Supelco BetaDex^® 225) after reversed phase (C18) solid-phase extraction. The limit of detection was 1 pg/ml for each enantiomer (approximately 500 fg on column) and the limit of quantification 5 pg/ml. The GC–PCI-MS method was applied for the quantification of tabun enantiomers in spiked swine blood after hemolysis and in spiked plasma of different species including humans.     

Direct injection of human plasma samples after ultrafiltration into programmed temperature vaporiser-gas chromatography–mass spectrometry with packed liner

The direct injection of plasma samples after ultrafiltration into a gas chromatograph using a packed injector liner was investigated. Ropivacaine, a local anaesthetic of the amide type and one of its metabolites (PPX) were used as model compounds in this evaluation. Phosphoric acid was added to the plasma to minimize the protein binding. After ultrafiltration, 50 μl of the sample was directly injected into the chromatographic system. No interfering peaks or damage to the GC or MS system were observed using ultrafiltration as a sample-preparation method. The validation of the method demonstrated good linearity and selectivity. The limits of quantification were 1.1 nM (301 pg/ml) and 1.4 nM (325 pg/ml) for ropivacaine and PPX, respectively. The liner had to be changed after 20 injections.     

Separation of ketoconazole enantiomers by chiral subcritical-fluid chromatography

The separation of ketoconazole enantiomers by subcritical-fluid chromatography using an amylose-based column is described. Drastic changes in the resolution have been obtained for the different organic modifiers evaluated, with ethanol providing the best results. Other chromatographic parameters such as temperature, pressure and flow-rate have also been studied. The best results in terms of resolution and analysis time were obtained using 30% ethanol (containing 0.1% triethylamine and 0.1% trifluoroacetic acid), a pressure of 300 bar, a temperature of 35 degrees C and a flow-rate of 3 ml/min. Under these conditions the ketoconazole enantiomers are resolved in a short time (less than 7 min) and with high resolution (4.29).     

A validated LC/MS/MS method for the quantification of pyrrole-2,3,5-tricarboxylic acid (PTCA), a eumelanin specific biomarker, in human skin punch biopsies

A novel skin tissue extraction method coupled with liquid chromatography-tandem mass spectrometry (LC/MS/MS) detection was developed and validated for the analysis of endogenous pyrrole-2,3,5-tricarboxylic acid (PTCA), a eumelanin specific biomarker, in human skin punch biopsies. The analyte is extracted from the matrix (2 mm skin punch biopsies) using a simple oxidative degradation procedure. The extract supernatants are evaporated, reconstituted in mobile phase solvent, and injected into the LC/MS/MS system without further derivatization. The chromatographic separation is achieved on a reverse phase high performance liquid chromatography (HPLC) column. The accuracy and precision of the method was determined over the concentration range of 1-1000 ng/mL PTCA from human skin extracts in three validation batch runs. Inter-assay precision (%CV) and accuracy (%R.E.) of the quality control samples were 相似文献   

Determination of tetrabromobisphenol A in human serum by liquid chromatography-electrospray ionization tandem mass spectrometry

A method for the determination of tetrabromobisphenol A (TBBPA) in human serum utilizing solid-phase extractions (SPEs) and liquid chromatography (LC) with electrospray ionization tandem MS (MS/MS) has been developed. After purification and concentration of TBBPA using consecutive SPEs on reversed-phase and normal-phase cartridges, the serum sample was subjected to LC. TBBPA was separated on a C18 reversed-phase column by gradient elution with a mixture of water, methanol, and acetonitrile as the mobile phase, and then detected with electrospray ionization MS/MS in negative ion mode. 13C12-TBBPA was suitable as an internal standard for the reproducible determination of TBBPA in human serum samples (5 g). The method has been validated in TBBPA concentration range of 5-100 pg per g serum, and the recoveries in the concentration range were higher than 83.3%. The repeatabilities of the proposed method of non-spiked control serum (6.3 pg per g serum) and spiked serum (added 5-100 pg per g serum) were within 10.0% as relative standard deviations. The limit of quantification (LOQ) for TBBPA was 4.1 pg per g serum, which was corresponded to 0.63 fmol on column.     

Determination of phloroglucinol in human plasma by high-performance liquid chromatography-mass spectrometry

A sensitive and selective liquid chromatographic method coupled with mass spectrometry (LC-MS) was developed for the quantification of phloroglucinol in human plasma. Resorcinol was used as internal standard, with plasma samples extracted using ethyl acetate. A centrifuged upper layer was then evaporated and reconstituted with mobile phase. The reconstituted samples were injected into a C(18) XTerra MS column (2.1 x 100 mm) with 3.5-microm particle size. The analytical column lasted for at least 500 injections. The mobile phase was 15% acetonitrile (pH 3.0), with flow-rate at 200 microl/min. The mass spectrometer was operated in negative ion mode with selective ion monitoring (SIM). Phloroglucinol was detected without severe interferences from plasma matrix when used negative ion mode. Phloroglucinol produced a parent molecule ([M-H](-)) at m/z 125 in negative ion mode. Detection of phloroglucinol in human plasma was accurate and precise, with quantification limit at 5 ng/ml. This method has been successfully applied to a study of phloroglucinol in human specimens.     

Direct cocktail analysis of drug discovery compounds in pooled plasma samples using liquid chromatography-tandem mass spectrometry

Direct plasma injection technology coupled with a LC-MS/MS assay provides fast and straightforward method development and greatly reduces the time for the tedious sample preparation procedures. In this work, a simple and sensitive bioanalytical method based on direct plasma injection using a single column high-performance liquid chromatography (HPLC) and tandem mass spectrometry (MS/MS) was developed for direct cocktail analysis of double-pooled mouse plasma samples for the quantitative determination of small molecules. The overall goal was to improve the throughput of the rapid pharmacokinetic (PK) screening process for early drug discovery candidates. Each pooled plasma sample was diluted with working solution containing internal standard and then directly injected into a polymer-coated mixed-function column for sample clean-up, enrichment and chromatographic separation. The apparent on-column recovery of six drug candidates in mouse plasma samples was greater than 90%. The single HPLC column was linked to either an atmospheric pressure chemical ionization (APCI) or electrospray ionization (ESI) source as a part of MS/MS system. The total run cycle time using single column direct injection methods can be achieved within 4 min per sample. The analytical results obtained by the described direct injection methods were comparable with those obtained by semi-automated protein precipitation methods within +/- 15%. The advantages and challenges of using direct single column LC-MS/MS methods with two ionization sources in combination of sample pooling technique are discussed.     

Detection of metabolites of lysergic acid diethylamide (LSD) in human urine specimens: 2-oxo-3-hydroxy-LSD,a prevalent metabolite of LSD

Seventy-four urine specimens previously found to contain lysergic acid diethylamide (LSD) by gas chromatography–mass spectrometry (GC–MS) were analyzed by a new procedure for the LSD metabolite 2-oxo-3-hydroxy-LSD (O-H-LSD) using a Finnigan LC–MS–MS system. This procedure proved to be less complex, shorter to perform and provides cleaner chromatographic characteristics than the method currently utilized by the Navy Drug Screening Laboratories for the extraction of LSD from urine by GC–MS. All of the specimens used in the study screened positive for LSD by radioimmunoassay (Roche Abuscreen^®). Analysis by GC–MS revealed detectable amounts of LSD in all of the specimens. In addition, isolysergic diethylamide (iso-LSD), a byproduct of LSD synthesis, was quantitated in 64 of the specimens. Utilizing the new LC–MS–MS method, low levels of N-desmethyl-LSD (nor-LSD), another identified LSD metabolite, were detected in some of the specimens. However, all 74 specimens contained O-H-LSD at significantly higher concentrations than LSD, iso-LSD, or nor-LSD alone. The O-H-LSD concentration ranged from 732 to 112 831 pg/ml (mean, 16 340 pg/ml) by quantification with an internal standard. The ratio of O-H-LSD to LSD ranged from 1.1 to 778.1 (mean, 42.9). The presence of O-H-LSD at substantially higher concentrations than LSD suggests that the analysis for O-H-LSD as the target analyte by employing LC–MS–MS will provide a much longer window of detection for the use of LSD than the analysis of the parent compound, LSD.     

Direct detection and quantification of 19-norandrosterone sulfate in human urine by liquid chromatography-linear ion trap mass spectrometry

19-Norandrosterone sulfate (19-NAS) is the sulfoconjugated form of 19-norandrosterone (19-NA), the major metabolite of the steroid nandrolone. A sensitive and accurate liquid chromatography/tandem mass spectrometry (LC-MS/MS) assay was developed for the direct measurement of 19-NAS in human urine samples. The method involved a quaternary amine SPE protocol and subsequently injection of the extract onto an analytical column (Uptisphere ODB, 150 mm x 3.0 mm, 5 microm) for chromatographic separation and mass spectrometry detection in negative electrospray ionisation mode. The sulfoconjugate of 19-NA was identified in urine by comparison of mass spectra and retention time with a reference substance. The limit of detection (LOD) and lowest limit of quantification (LLOQ) of 19-NAS were of 40 pg/mL and 200 pg/mL, respectively. For a nominal concentration of 2 ng/mL, recovery (94%), intra-day precision (2.7%), intra-assay precision (6.6%) and inter-assay precision (14.3%) were determined. Finally, this analytical method was applied for quantifying the concentration of 19-NAS in doping samples, using calibration curves (0.2-20 ng/mL) and the standard-addition method. The results show the feasibility of applying this LC-MS/MS assay as a complementary tool to detect misuse of nandrolone or nandrolone precursors.     

Determination of picogram levels of heptylphysostigmine in human plasma using high-performance liquid chromatography with fluorescence detection

A sensitive (50 pg/ml) method for the determination of heptylphysostigmine in human plasma is described. The procedure is based on liquid—liquid extraction of the drug from buffered plasma, and analysis of the concentrated organic extract using high-performance liquid chromatography on a silica column, under normal-phase chromatographic conditions, with fluorescence detection. Physostigmine was used as an internal standard. The assay has been fully validated in the concentration range 50–2000 pg/ml and utilized for the analysis of clinical samples from subjects dosed with heptylphysostigmine.     

High performance analysis of the cyanobacterial metabolism via liquid chromatography coupled to a LTQ-Orbitrap mass spectrometer: evidence that glucose reprograms the whole carbon metabolism and triggers oxidative stress

Cyanobacteria are environmentally important photosynthetic microorganisms attracting a growing attention in various areas of basic and applied researches. To better understand their metabolism, we presently report on the development of a robust and simple protocol for facile extraction and high throughput analysis of the metabolites of the widely-used strain Synechocystis PCC6803 through liquid chromatography coupled to high resolution mass spectrometry (LC/MS). Our analytical method was developed and tested with 102 reference compounds representative of the chemical diversity of polar cell metabolites, and Synechocystis cell extracts spiked with 37 reference compounds. These samples were analyzed with two chromatographic systems, each coupled to a LTQ-Orbitrap mass spectrometer: a liquid chromatographic system equipped with a pentafluorophenylpropyl column (the PFPP-LC/MS system), and an ultra-high performance liquid chromatographic system with a C₁₈-reversed phase column (the C₁₈-UHPLC/MS system). We showed that the PFPP-LC/MS method performs better than the C₁₈-UHPLC/MS method in terms of retention, separation and detection of metabolites. Consequently, we applied the PFPP-LC/MS method to analyze the metabolome of Synechocystis growing under various conditions of light and glucose, which strongly influence cell growth. We found that glucose increases glucose storage (synthesis of glycogen-like polysaccharide) and catabolism (oxidative pentose phosphate pathway and glycolysis), while it decreases the Calvin–Benson cycle that consumes photosynthetic electrons for CO₂ assimilation. Depending on light and glucose availabilities, this global metabolic reprogramming can generate an oxidative stress, likely through the recombination of the glucose-spared electrons with the photosynthetic oxygen thereby producing toxic reactive oxygen species.     

Detection of testosterone propionate administration in horse hair samples

A sensitive and specific method has been developed to detect semi-quantitatively testosterone in horse hair samples. The method involved a washing step with sodium dodecylsulfate aqueous solution. The mane and tail hair samples (100mg) were dissolved in 1 mL of sodium hydroxide for 15 min at 95 degrees C in the presence of d3-boldenone used as internal standard. The next three steps involved diethyl ether extraction and a solid phase extraction on Isolute C18 (EC) cartridges eluted with methanol. The residue was derivatized by adding 100 microL of acetonitrile and 30 microL of PFPA then incubating for 15 min at 60 degrees C. After evaporation, 30 microL of hexane was added and 2.5 microL was injected into the column (a bonded phase fused silica capillary column DB5MS, 30 m x 0.25 mm i.d. x 0.25 microm film thickness) of a Trace GC chromatograph. In order to improve the sensitivity of the method, damping gas flow has been optimized. Testosterone was identified in MS(2) full scan mode on the Polaris Q instrument. The assay was capable of detecting less than 1 pg mg(-1). The recovery was close to 90%. The analysis of tail and mane samples collected from a gelding horse having received a single dose of testosterone propionate (1 mg kg(-1)) showed the presence of testosterone in the range of 1-6 pg mg(-1) in hair collected during 5 months after administration.     

Estimation of amniotic fluid phospholipids by high-performance liquid chromatography

We have developed a high-performance liquid chromatographic (HPLC) method for the analyses of surface-active amniotic fluid phospholipids, lecithin (L), sphingomyelin (S), phosphatidyl glycerol (PG), phosphatidyl inositol (PI), phosphatidyl ethanolamine (PE), and phosphatidyl serine (PS), which are important in the prediction of fetal lung maturity. The method incorporates an internal standard in the amniotic fluid extract, and utilizes a 10-μl aliquot of a 2:1 chloroform—methanol extract of amniotic fluid injected onto a 5-μm DIOL or CN HPLC column, and a variable-wavelength detector set at 203 nm.Amniotic fluid phospholipid estimations were determined on 40 amniotic fluid samples by the HPLC method and by the routine thin-layer chromatographic (TLC) method. Good agreement was observed between the two methods for the L/S ratio, PG, and PI (r_PG 0.94, r_PI 0.95, r_L/S 0.97).The advantages of the HPLC procedure include: (i) Selective separation for PG, PI, PS, and PE, as well as L and S at the same time. (ii) The internal standard allows individual concentration of phospholipids to be estimated. (iii) The procedure is rapid: 16 min for a single assay compared with 50 min for the standard TLC procedure.     

Simultaneous determination of global DNA methylation and hydroxymethylation levels by hydrophilic interaction liquid chromatography-tandem mass spectrometry

Methylation of DNA at the 5-position of cytosine (Cyt) is a well-studied epigenetic pathway implicated in gene silencing and embryogenesis. Recently, in addition to 5-methylcytosine (5mC), substantial amounts of 5-hydroxymethylcytosine (5hmC) have been detected in certain mammalian tissues. Here, we developed and validated a hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS) method for the simultaneous determination of Cyt, 5mC, and 5hmC levels in biological samples. DNA was extracted with phenol-chloroform, hydrolyzed using 88% formic acid at 140 °C, separated using a bridged ethylene hybrid HILIC column, and analyzed by tandem MS. The linearity was established over the concentration range of 1 to 500 ng/mL for Cyt, 0.2 to 100 ng/mL for 5mC, and 0.1 to 50 ng/mL for 5hmC, and the correlation coefficients were all >0.99. Limits of detection were 1 pg/mL for Cyt, 45 pg/mL for 5mC, and 57 pg/mL for 5hmC, and the limit of quantification values for Cyt, 5mC, and 5hmC were 2 pg/mL, 90 pg/mL, and 100 pg/mL, respectively. The relative standard deviation (RSD) of the intraday precision ranged from 1.87% to 4.84% and the interday precision from 2.69% to 4.98%. The recovery of the method varied from 88.25% to 104.39%. The method was then applied to the analysis of DNA from biological samples, establishing its potential for helping researchers understand the roles of modified nucleobases in DNA.     

Evaluation of reductive amperometric detection in the liquid chromatographic determination of antineoplastic platinum complexes

The usefulness of reductive electrochemical detection at mercury drop electrodes has been determined for platinum complexes separated by solvent-generated anion-exchange high-performance liquid chromatography. Both current-sampled dropping mercury and hanging mercury drop electrodes (DME and HMDE) provide significant advantages over UV absorbance and off-line non-flame atomic absorption detection. The effects of chromatographic and polarographic parameters on analytical system performance have been investigated. By raising the detector cell temperature, the detector response to cis-dichlorodiammineplatinum(II) (DDP) can be shifted anodically to 0.0 V vs. Ag/AgCl, thereby increasing detector selectivity for this compound. The noise-limited minimum detectable quantities of DDP with DME and HMDE are 1.8 ng and 70 pg injected, respectively. DDP can be determined in untreated urine at levels below 100 ng/ml.