Determination of glimepiride in human plasma by liquid chromatography-electrospray ionization tandem mass spectrometry

A sensitive and specific high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS-MS) method has been developed at our center for the determination of glimepiride in human plasma. After the addition of the internal standard, plasma samples were extracted by liquid-liquid extraction technique using diethyl ether. The compounds were separated on a prepacked C18 column using a mixture of acetonitrile, methanol and ammonium acetate buffer as mobile phase. A Finnigan LCQDUO ion trap mass spectrometer connected to an Alliance Waters HPLC was used to develop and validate the method. The analytical method was validated according to the FDA bioanalytical method validation guidance. The results were within the accepted criteria as stated in the aforementioned guidance. The method was proved to be sensitive and specific by testing six different plasma batches. Linearity was established for the range of concentrations 5.0-500.0 ng/ml with a coefficient of determination (r2) of 0.9998. Accuracy for glimepiride ranged from 100.58 to 104.48% at low, mid and high levels. The intra-day precision was better than 12.24%. The lower limit of quantitation (LLOQ) was identifiable and reproducible at 5.0 ng/ml with a precision of 7.96%. The proposed method enables the unambiguous identification and quantitation of glimepiride for pharmacokinetic, bioavailability or bioequivalence studies.     

Determination of nitrofuran metabolites in milk by liquid chromatography-electrospray ionization tandem mass spectrometry

An LC-ESI-MS-MS method for the analysis of metabolites of four nitrofurans (furazolidone, furaltadone, nitrofurazone and nitrofurantoin) in raw milk has been developed. The samples were achieved by hydrolysis of the protein-bound drug metabolites, derivatization with 2-nitrobenzaldehyd (2-NBA) and clean-up extraction liquid-liquid with ethyl acetate. LC separation was achieved by using a Phenomenex Luna C-18 column. The mass spectrometer operated in multiple reaction monitoring mode (MRM) with positive electro-spray interface (ESI). The method validation was done according to the criteria laid down in Commission Decision No. 2002/657 EC. The validation includes the determination of linearity, repeatability, within-laboratory reproducibility, accuracy, decision limit (CCalpha) and detection capability (CCbeta). The calibration curves were linear, with typical (R(2)) values higher than 0.991. The coefficient of variation (CV, %) was lower than 9.3% and the accuracy (RE, %) ranged from -9.0% to 7.0%. CV within-laboratory reproducibility was lower than 13%. The limits of decision (CCalpha) and detection capability (CCbeta) were 0.12-0.29 microg/kg and 0.15-0.37 microg/kg, thus below the minimum required performance limit (MRPL) set at 1 microg/kg by the UE. This validated method was successfully applied for the determination of nitrofuran metabolites in a large number of milk samples.     

Determination of proteins in infant formula by high-performance liquid chromatography-electrospray tandem mass spectrometry

To determine the protein content of formula, gel electrophoresis was performed on the infant formula samples and the entire protein patterns were analyzed by nano-high performance liquid chromatography-electrospray tandem mass spectrometry (nano-HPLC/ESI/MS/MS). From the commercial infant formula profiled in this study, a total of 154 peptides, corresponding to 31 unique proteins were identified by nano-HPLC/ESI/MS/MS. Each of the identified peptides was reconfirmed by a strict integrated approach using tandem mass spectra. This protein profiling method using gel electrophoresis coupled with nano-HPLC/ESI/MS/MS and manual evaluation is a sensitive and accurate method for protein identification as well as a powerful tool for monitoring various types of food products.     

Determination of cortisol in human saliva using liquid chromatography-electrospray tandem mass spectrometry

The aim of this work was to develop a method for determination of cortisol in saliva by liquid chromatography-tandem mass spectrometry (LC-MS-MS). Saliva was sampled on Salivette tubes. These were centrifuged, deuterium-labeled cortisol was added as internal standard and the proteins precipitated by acetonitrile. The supernatant was evaporated, dissolved in methanol acidified with acetic acid and analyzed by LC-MS-MS. The with-in run precision, tested by pooling saliva samples from volunteers and then analyzing these in a single run, was found to be 7% at 0.7 microgram l(-1). The between-run precision was tested by analysis of the same samples at different days and found to be 11% at 2.5 microgram l(-1). The limit of quantification was 0.5 microgram l(-1). The method was applied for analysis of saliva samples from three volunteers during their last week before vacation and the first and second week on vacation. In addition, the method was compared to analysis by an immunological method. The values from the immunological method were 2.7 times higher than the LC-MS-MS results.     

Determination of tacrolimus in rabbit aqueous humor by liquid chromatography-electrospray ionization tandem mass spectrometry

A simple, sensitive and selective liquid chromatographic-tandem mass spectrometric (LC-MS/MS) method was developed for the determination of tacrolimus (FK506) in rabbit aqueous humor. After a simple protein-precipitation by methanol, the post-treatment samples were separated on a reversed-phase, Thermo-Hypersil-BDS-C18 column with a mobile phase of a mixture of 0.1% formic acid in water, methanol and acetonitrile (5:85:10, v/v/v). Tacrolimus and ritonavir (internal standard, IS) were all detected by the selected reaction-monitoring (SRM) mode. The method developed was validated in rabbit aqueous humor with a daily working range of 0.5-100 ng/ml with correlation coefficient, r>0.99 and a sensitivity of 0.5 ng/ml as lower limit of quantification, respectively. This method was fully validated for the accuracy, precision, possible matrix effect and stability. The method proved to be accurate and specific, and was applied to the pharmacokinetic study of tacrolimus in rabbit aqueous humor.     

Determination of bulleyaconitine A in human plasma by liquid chromatography-electrospray ionization tandem mass spectrometry

A sensitive and specific high-performance liquid chromatography (HPLC)-electrospray ionization tandem mass spectrometry (MS-MS) was developed for the determination of bulleyaconitine A (BLA) in human plasma. BLA and internal standard (I.S.) ketoconazole were extracted from the plasma by a liquid-liquid extraction. The supernatant was evaporated to complete dryness and reconstituted with acetonitrile containing 0.1% acetic acid before injecting into an ODS MS column. The gradient mobile phase was composed of a mixture of acetonitrile (containing 0.1% acetic acid, v/v) and 0.1% acetic acid aqueous solution eluted at 0.3 ml/min. BLA and I.S. were determined by multiple reaction monitoring using precursor-->product ion combinations at m/z 644.6-->584.3 and 531.2-->81.6, respectively. Linearity was established for the concentration range of 0.12-6 ng/ml. The recoveries of BLA ranged from 96.93 to 113.9% and the R.S.D. was within 20%. The method is rapid and applicable to the pharmacokinetic studies of BLA in human.     

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 the active metabolite of sibutramine by liquid chromatography-electrospray ionization tandem mass spectrometry

A sensitive and specific method for the determination of the active primary amine metabolite of sibutramine, N-di-desmethylsibutramine (BTS 54,505), in human plasma was developed, based on high-performance liquid chromatography (HPLC)-electrospray ionization tandem mass spectrometry (MS-MS). The samples were extracted from plasma with methyl tert.-butyl ether, followed by separation and evaporation after addition of the internal standard, propranolol, and basification with sodium hydroxide. The residue was reconstituted in mobile phase and injected into the HPLC-MS-MS system. Chromatography was performed on an ODS MS column with a mobile phase consisting of acetonitrile (containing 0.1% trifluoroacetic acid, v/v)-0.1% trifluoroacetic acid (55:45, v/v) at a flow-rate of 0.3 ml/min. Multiple reaction monitoring using precursor-->product ion combinations at m/z 252.00-->125.00 and 260.00-->115.70 was applied to determine BTS 54,505 and propranolol, respectively. Linearity was confirmed in the concentration range 0.328-32.8 ng/ml in human plasma and the imprecision of this assay was less than 19.90% over the entire concentration range. The method is sufficiently sensitive and repeatable to be used in pharmacokinetic studies.     

Lisinopril quantification in human plasma by liquid chromatography-electrospray tandem mass spectrometry

An analytical method based on liquid chromatography with positive ion electrospray ionization (ESI) coupled to tandem mass spectrometry detection was developed for the determination of Lisinopril in human plasma using Enalaprilat as internal standard. The analyte and internal standard were extracted from the plasma samples by solid-phase extraction using Waters HLB Oasis SPE cartridges and chromatographed on a C8 analytical column. The mobile phase consisted of acetonitrile/water (60:40, v/v) + 20 mM acetic acid + 4.3 mM of triethylamine. The method had a chromatographic total run-time of 6.5 min and was linear within the range 2.00-200 ng/ml. Detection was carried out on a Micromass triple quadrupole tandem mass spectrometer by multiple reaction monitoring (MRM). The precision (CV%) and accuracy, calculated from limit of quantification (LOQ) samples (n = 8), were 8.9 and 98.9%, respectively. The method herein described was employed in a bioequivalence study of two tablet formulations of Lisinopril 20mg.     

Determination of glycemic monitoring marker 1,5-anhydroglucitol in plasma by liquid chromatography-electrospray tandem mass spectrometry

Previous studies have shown that plasma 1,5-anhydroglucitol (1,5-AG) is markedly reduced among diabetic patients and therefore serves as a sensitive marker for short-term glycemic control. The current study describes the development of the liquid chromatography negative ion electrospray tandem mass spectrometry (LC-MS/MS) method to measure 1,5-AG in human plasma. The samples were pre-treated with protein precipitation and an isotope-labeled internal standard was used. Chromatographic separation was achieved on amide column (150 mm x 2.0mm i.d., 5 microm) followed by detection with multiple reaction monitoring mode. Linearity, accuracy, precision, recovery, matrix effect, and stability were evaluated during method validation over the range of 1-50 microg/mL. The validated method has been clinically applied among 159 type 2 diabetic patients and 290 control subjects. A marked reduction in 1,5-AG levels among the diabetic patients and significant between-gender difference in nondiabetic subjects were observed.     

Lansoprazole quantification in human plasma by liquid chromatography-electrospray tandem mass spectrometry

An analytical method based on liquid chromatography with positive ion electrospray ionization (ESI) coupled to tandem mass spectrometry detection was developed for the determination of lansoprazole in human plasma using omeprazole as the internal standard. The analyte and internal standard were extracted from the plasma samples by liquid-liquid extraction using diethyl-ether-dichloromethane (70:30; v/v) and chromatographed on a C(18) analytical column. The mobile phase consisted of acetonitrile-water (90:10; v/v)+10 mM formic acid. The method has a chromatographic total run time of 5 min and was linear within the range 2.5-2000 ng/ml. Detection was carried out on a Micromass triple quadrupole tandem mass spectrometer by Multiple Reaction Monitoring (MRM). The intra- and inter-run precision, calculated from quality control (QC) samples, was less than 3.4%. The accuracy as determined from QC samples was less than 9%. The method herein described was employed in a bioequivalence study of two capsule formulations of lansoprazole.     

Determination of fetal bile acids in biological fluids from neonates by gas chromatography-negative ion chemical ionization mass spectrometry

A method has been developed for microanalysis of fetal bile acids in biological fluids from neonates by capillary gas chromatography-mass spectrometry using negative-ion chemical ionization of pentafluorobenzyl ester-dimethylethylsilyl ether derivatives of bile acids. Calibration curves for the bile acid derivatives are useful over the range 0.1–100 pg and the detection limit for bile acids was 1 fg (S/N=5) using isobutane as a reagent gas. Recoveries of the bile acids and their glycine and taurine conjugates from bile acid-free serum and dried blood discs ranged from 92 to 101% and from 93 to 108%, respectively, of the added amounts of their standard samples. The analysis of bile acids on a dried blood disc, meconium and urine from infants, exhibited significant hydroxylation at the 1β-, 2β-, 4β and 6α-positions of the usual bile acids, cholic and chenodeoxycholic acids, for the urinary or fecal excretion of bile acids in the fetal and neonatal periods. The present method was applied clinically to analyze bile acids on a dried blood disc from neonatal patients with congenital biliary atresia and hyper-bile-acidemia.     

High-performance liquid chromatographic-electrospray mass spectrometric analysis of bile acids in biological fluids

The present work describes the development of HPLC-mass spectrometric systems equipped with an electrospray interface for the quantitative analysis of bile acids. Good separation of free as well as glycine- and taurine-conjugated bile acids was achieved with a C₁₈ reversed-phase column (3 μm particle size, 70 × 4.6 mm I.D.) employing methanol-15 mM ammonium acetate as the mobile phase for both isocratic and gradient mode, at a flow-rate of 0.3 ml/min. This system permits post-column splitting of the eluate for analysis by two different detectors: (1) electrospray-mass spectrometer with a flow-rate of 18 μl/min; and (2) a complementary evaporative light scattering mass detector. When bile salts were ionized in the electrospray interface operating in the negative-ion mode, only [M  H]⁻ molecular ions were generated; the detection limit was 15 pg injected for all bile acids studied. In the second system, a semi-micro pre-column splitting apparatus (Acurate, LC Packings) was utilized: with this device the flow-rate from the HPLC pump was reduced to 1.4 μl/min and bile acids were separated with a micro-bore C₁₈ column (3 μm particle size, 150 × 0.30 I.D.), using the same mobile phase as above. With this latter system, a head-column enrichment technique can be used: the amount injected can be increased from 60 to 200 nl, permitting an improvement in the detection limit to 5 pg injected. Application of the HPLC-electrospray-mass spectrometric method to bile and serum bile acid analysis is described; preliminary data on the ability of the first system to determine the ¹³C/¹²C isotope ratio in ¹³C-labeled bile acid enriched serum is also critically discussed.     

Determination of long-chain fatty acid acyl-coenzyme A compounds using liquid chromatography-electrospray ionization tandem mass spectrometry

Acyl-CoAs have important role in fat and glucose metabolism of the cells. In this study we have developed an on-line HPLC-ESI-MS/MS method for determination of long-chain acyl-CoA compounds in rat liver samples. Six long-chain acyl-CoAs (C16:0, C16:1, C18:0, C18:1, C20:0 and C20:4) were separated with a C4 reversed-phase column using triethylamine acetate and acetonitrile gradient. Negative electrospray ionization is very suitable for acyl-CoA compounds and excellent MS/MS spectra for long-chain acyl-CoAs can be obtained. MS/MS method with an ion trap mass spectrometer makes it possible to identify and quantitate individual acyl-CoAs simultaneously. The method proved to be sensitive enough for determination of all compounds of interest using 0.4-0.7 g of tissue and was validated in the range of 0.1-15.0 pmol/microl.     

Determination of levocetirizine in human plasma by liquid chromatography-electrospray tandem mass spectrometry: application to a bioequivalence study

We describe a liquid chromatography-tandem mass spectrometric method (LC-MS/MS) for levocetirizine quantification (I) in human plasma. Sample preparation was made using a fexofenadine (II) addition as internal standard (IS), liquid-liquid extraction using cold dichloromethane, and dissolving the final extract in acetonitrile. I and II (IS) were injected in a C18 column and the mobile phase composed of acetonitrile:water:formic acid (80.00:19.90:0.10, v/v/v) and monitored using positive electrospray source with tandem mass spectrometry analyses. The selected reaction monitoring (SRM) was set using precursor ion and product ion combinations of m/z 389>201 for I and m/z 502>467 for II. The limit of quantification and the dynamic range achieved were 0.5ng/mL and 0.5-500.0ng/mL. Validation results on linearity, specificity, accuracy, precision and stability, as well as its application to the analysis of plasma samples taken up to 48h after oral administration of 5mg of levocetirizine dichloridrate in healthy volunteers demonstrate its applicability to bioavailability studies.     

Determination of hexaconazole in surface water samples from river and the sea by liquid chromatography-electrospray tandem mass spectrometry

A simple and selective method was developed for determining the concentration of hexaconazole in river and sea water samples by using liquid chromatography/tandem mass spectrometry with an electrospray ionization interface in the positive ion mode and selective reaction monitoring mode. Trace amounts of hexaconazole were collected in a Sep-Pak Plus tC18 cartridge that was eluted with methanol. The detection limit for hexaconazole was 6 ng/l. The recovery of a standard aqueous solution containing 1 microg/l was 96%. The recovery of hexaconazole in the river and sea water samples was 95% and 90%, respectively. Hexaconazole was not detected in the sea water samples. Trace peaks of hexaconazole were found in the river water samples, the concentration being less than 6 ng/l in all cases. The biological degradation of hexaconazole was tested by using river water. No degradation of hexaconazole was apparent in river water incubated at 20 degrees C for 3 weeks.     

Determination of memantine in human plasma by liquid chromatography-electrospray tandem mass spectrometry: application to a bioequivalence study

A simple, sensitive and specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantification of memantine (I) in human plasma is presented. Sample preparation consisted of the addition of amantadine (II) as internal standard (IS), liquid-liquid extraction in basic conditions using a mixture of diethyl ether-chloroform (7:3, v/v) as extracting solvent, followed by centrifugation, solvent evaporation and sample reconstitution in methanol. Both I and II (internal standard) were analyzed using a C18 column and a mobile phase composed of methanol-water-formic acid (80:20:0.1, v/v/v). Eluted compounds were monitored using positive mode electrospray (ES) tandem mass spectrometry. The analyses were carried out by selected reaction monitoring (SRM) using the parent to daughter combinations of m/z 180>163 (memantine) and m/z 152>135 (amantadine). The peak areas from the analyte and IS were used for quantification of I. The achieved limit of quantification (LOQ) was 0.1 ng/mL; the assay exhibited a linear dynamic range of 0.1-50.0 ng/mL with a determination coefficient (r2) of at least 0.98. Validation results on linearity, specificity, accuracy, precision and stability, as well as on application to the analysis of samples taken up to 320 h after oral administration of 20mg (two 10mg capsules) of I in healthy volunteers demonstrated the applicability to bioequivalence studies.     

Cytokinin profiling in plant tissues using ultra-performance liquid chromatography-electrospray tandem mass spectrometry

We have developed a simple, high-throughput batch immunoextraction (IAE) micropurification procedure for extracting a wide range of naturally occurring cytokinins (bases, ribosides, O- and N-glucosides, and nucleotides) from plant tissues in solutions that are compatible with ultra-performance liquid chromatography (UPLC), thereby facilitating sensitive subsequent analysis. The UPLC system was coupled to a tandem quadrupole mass spectrometer (MS/MS) equipped with an electrospray interface (ESI). Small (mg) amounts of tissues were purified by solid-phase extraction (SPE) followed by an immunoaffinity clean-up step and two fast chromatographic separations of most cytokinin metabolites (bases, ribosides, and 9-glucosides in the first, O-glucosides and nucleotides in the second). Using UPLC, the runs were up to 4-fold faster than in standard cytokinin analyses, and both retention times and injection volumes were less variable (RSDs, 0.15-0.3% and 1.0-5.5%, respectively). In multiple reaction monitoring (MRM) mode, the detection limit for most of the cytokinins analyzed was close to 1 fmol (5-25 fmol for O-glucosides and nucleotides) and the linear range spanned at least five orders of magnitude. The extraction and purification method was optimized using poplar (Populus × canadensis Moench, cv Robusta) leaf samples, and the analytical accuracy was further validated using IAE-purified 10-day-old Arabidopsis thaliana plants spiked with 1 and 10 pmol of cytokinin derivatives. This approach can be used for rapid, sensitive qualitative and/or quantitative analysis of more than 50 natural cytokinins in minute amounts of plant tissues with high performance, robustness, and accuracy.     

Quantitation of fatty acyl-coenzyme As in mammalian cells by liquid chromatography-electrospray ionization tandem mass spectrometry

Fatty acyl-CoAs participate in numerous cellular processes. This article describes a method for the quantitation of subpicomole amounts of long-chain and very-long-chain fatty acyl-CoAs by reverse-phase LC combined with electrospray ionization tandem mass spectrometry in positive ion mode with odd-chain-length fatty acyl-CoAs as internal standards. This method is applicable to a wide range of species [at least myristoyl- (C14:0-) to cerotoyl- (C26:0-) CoA] in modest numbers of cells in culture ( approximately 10(6)-10(7)), with analyses of RAW264.7 cells and MCF7 cells given as examples. Analysis of these cells revealed large differences in fatty acyl-CoA amounts (12 +/- 1.0 pmol/10(6) RAW264.7 cells vs. 80.4 +/- 6.1 pmol/10(6) MCF7 cells) and subspecies distribution. Very-long-chain fatty acyl-CoAs with alkyl chain lengths > C20 constitute <10% of the total fatty acyl-CoAs of RAW264.7 cells versus >50% for MCF7 cells, which somewhat astonishingly contain approximately as much C24:0- and C26:0-CoAs as C16:0- and C18:0-CoAs and essentially equal amounts of C26:1- and C18:1-CoAs. This simple and robust method should facilitate the inclusion of this family of compounds in "lipidomics" and "metabolomics" studies.     

Analysis of glycerophosphoinositol by liquid chromatography-electrospray ionisation tandem mass spectrometry using a beta-cyclodextrin-bonded column

Glycerophosphoinositol (GroPIns) has been demonstrated to have important roles in many intracellular regulatory processes. GroPIns has been analysed for many years by anion-exchange HPLC after radiolabelling of cells in culture, but no method has been developed, to our knowledge, for the direct detection and quantitation of the unlabelled compound in such biological samples. Here is reported a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the direct quantitative analysis of GroPIns that can indeed be applied to cell extracts. Analyses were performed on a beta-cyclodextrin-bonded HPLC column using a binary mobile phase of acetonitrile and 20 mM ammonium formate in water, which allowed direct on-line detection by tandem mass spectrometry in negative electrospray ionisation (ESI) mode. The method was applied to the quantitative analysis of GroPIns in selected rat cell lines after a two-phase acid extraction of cultured cells using external calibration. The potential matrix signal suppression effects were investigated by the parallel quantitation of GroPIns in extracts of selected cultured cell lines with both external calibration and the standard additions method. The accuracy data obtained demonstrated the feasibility of external calibration, so allowing a simpler and less time-consuming approach than that of the standard additions method.