Quantification of free and total sialic acid excretion by LC-MS/MS

BACKGROUND: The main purpose for measuring urinary free sialic acid (FSA) is to diagnose sialic acid (SA) storage diseases. Elevated amounts of conjugated sialic acid (CSA) are observed in several diseases indicating the need to quantify CSA as well. A LC-MS/MS method for quantification of FSA and total sialic acid (TSA) in urine is developed and validated. METHODS: FSA is analyzed directly after filtration of urine samples. For determination of TSA an enzymatic (neuraminidase) and a chemical (acid) hydrolysis were compared. 13C3-sialic acid was used as internal standard. LC-MS/MS was performed in negative electrospray ionisation mode with multiple reaction monitoring of transitions m/z 308.2-->87.0 (SA) and m/z 311.2-->90.0 (13C3-SA). CSA was calculated by subtracting FSA from TSA. RESULTS: Limit of detection for FSA and TSA was 0.3 and 1.7 micromol/L, respectively. Limit of quantification for FSA and TSA was 1.0 and 5.0 micromol/L. Intra- and inter-assay variations of FSA were 4.6% and 6.6% (n=10) for FSA and 6.5% and 3.6% (n=10) for TSA. Linearity was tested till 7800 micromol/L (r2=0.9998). Values of SA analyzed after neuraminidase- or acid hydrolysis treatment were comparable. Urine samples from patients with inborn errors of SA (related) metabolism were analyzed and compared with age-related reference values. CONCLUSION: A method has been developed for routine determination of urinary FSA and TSA. The method is rapid, specific, robust and sensitive. Age-related reference values for FSA, TSA and CSA were determined and improved diagnostic efficacy.     

Liquid chromatography-tandem mass spectrometric determination of ceramides and related lipid species in cellular extracts

A liquid chromatography-electrospray ionization tandem mass spectrometric (LC-MS/MS) method was developed for the simultaneous qualitative and quantitative determination of sphingolipid metabolites such as ceramides, sphingisine, sphinganine, sphingomyelins, and ceramide 1-phosphates in the extracts of human promyelocytic leukemia cells (HL-60). The assay uses C(4) ceramide as an internal standard; simple liquid extraction; a short XTerra MS C(18) (3 microm, 50 mm x2.0 mm) column; a gradient mobile phase of 5mM ammonium formate (pH 4.0)/methanol/tetrahydrofuran (5/2/3-->1/2/7); mass spectrometric detection using electrospray ionization. This LC-MS/MS method allowed the identification of 22 sphingolipid derivatives at pmol levels. In addition, this technique was successfully applied to analyze the changes of the sphingolipids profiles in cancer cells treated with apoptosis inducing agents, C(2) ceramide and H(2)O(2).     

Determination of amiodarone and desethylamiodarone in horse plasma and urine by high-performance liquid chromatography combined with UV detection and electrospray ionization mass spectrometry

A rapid method for the quantification of amiodarone and desethylamiodarone in animal plasma using high-performance liquid chromatography combined with UV detection (HPLC-UV) is presented. The sample preparation includes a simple deproteinisation step with acetonitrile. In addition, a sensitive method for the quantification of amiodarone and desethylamiodarone in horse plasma and urine using high-performance liquid chromatography combined with electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) is described. The sample preparation includes a solid-phase extraction (SPE) with a SCX column. Tamoxifen is used as an internal standard for both chromatographic methods. Chromatographic separation is achieved on an ODS Hypersil column using isocratic elution with 0.01% diethylamine and acetonitrile as mobile phase for the HPLC-UV method and with 0.1% formic acid and acetonitrile as mobile phase for the LC-MS/MS method. For the HPLC-UV method, good linearity was observed in the range 0-5 microg ml(-1), and in the range 0-1 microg ml(-1) for the LC-MS/MS method. The limit of quantification (LOQ) was set at 50 and 5 ng ml(-1) for the HPLC-UV method and the LC-MS/MS method, respectively. For the UV method, the limit of detection (LOD) was 15 and 10 ng ml(-1) for amiodarone and desethylamiodarone, respectively. The LODs of the LC-MS/MS method in plasma were much lower, i.e. 0.10 and 0.04 ng ml(-1) for amiodarone and desethylamiodarone, respectively. The LODs obtained for the urine samples were 0.16 and 0.09 ng ml(-1) for amiodarone and desethylamiodarone, respectively. The methods were shown to be of use in horses. The rapid HPLC-UV method was used for therapeutic drug monitoring after amiodarone treatment, while the LC-MS/MS method showed its applicability for single dose pharmacokinetic studies.     

Quantitative analysis of N-terminal valine peptide adducts specific for 1,2-epoxy-3-butene

Butadiene (BD) metabolism shows gender, species and concentration dependency, making the extrapolation of animal results to humans complex. BD is metabolized mainly by cytochrome P450 2E1 to three epoxides, 1,2-epoxy-3-butene (EB), 1,2;3,4-diepoxybutane (DEB) and 1,2-epoxy-butanediol (EB-diol). For accurate risk assessment it is important to elucidate species differences in the internal formation of the individual epoxides in order to assign the relative risks associated with their different mutagenic potencies. Analysis of N-terminal globin adducts is a common approach for monitoring the internal formation of BD derived epoxides. Our long term strategy is to develop an LC-MS/MS method for simultaneous detection of all three BD hemoglobin adducts. This approach is modeled after the recently reported immunoaffinity LC-MS/MS method for the cyclic N,N-(2,3-dihydroxy-1,4-butadyil)-valine (pyr-Val, derived from DEB). We report herein the analysis of the EB-derived 2-hydroxyl-3-butenyl-valine peptide (HB-Val). The procedure utilizes trypsin hydrolysis of globin and immunoaffinity (IA) purification of alkylated heptapeptides. Quantitation is based on LC-MS/MS monitoring of the transition from the singly charged molecular ion of HB-Val (1-7) to the a(1) fragment. Human HB-Val (1-11) was synthesized and used for antibody production. As internal standard, the labeled rat-[(13)C(5)(15)N]-Val (1-11) was prepared through direct alkylation of the corresponding peptide with EB. Standards were characterized and quantified by LC-MS/MS and LC-UV. The method was validated with different amounts of human HB-Val standard. The recovery was >75% and coefficient of variation <25%. The LOQ was set to 100 fmol/injection. For a proof of principal experiment, globin samples from male and female rats exposed to 1000 ppm BD for 90 days were analyzed. The amounts of HB-Val present were 268.2+/-56 and 350+/-70 pmol/g (mean+/-S.D.) for males and females, respectively. No HB-Val was detected in controls. These data are much lower compared to previously reported values measured by GC-MS/MS. The difference may be due higher specificity of the LC-MS/MS method to the N-terminal peptide from the alpha-chain versus derivatization of both alpha- and beta-chain by Edman degradation, and possible instability of HB-Val adducts during long term storage (about 10 years) between the analyses. These differences will be resolved by examining recently collected samples, using the same internal standard for parallel analysis by GC-MS/MS and LC-MS/MS. Based on our experience with pyr-Val adduct assay we anticipate that this assay will be suitable for evaluation of HB-Val in multiple species.     

A GC/MS validated method for the nanomolar range determination of succinylacetone in amniotic fluid and plasma: an analytical tool for tyrosinemia type I

A sensitive and accurate stable isotope dilution GC/MS assay was developed and validated for the quantification of succinylacetone (SA) in plasma and amniotic fluid (AF). SA is pathognonomic for tyrosinemia type I, a genetic disorder caused by a reduced activity of fumarylacetoacetate hydrolase (FAH). In untreated patients, SA can easily be measured in plasma and urine because the expected concentrations are in the micromol/L range. Due to a founder effect, the province of Quebec has an unusually high prevalence of tyrosinemia type I, hence, the quantification of SA in AF or plasma of treated patients in the nmol/L range becomes very useful. The method utilizes 13C5-SA as an internal standard and a three-step sample treatment consisting of oximation, solvent extraction and TMCS derivatization. The assay was validated by recording the ion intensities of m/z 620 for SA and m/z 625 for ISTD in order to demonstrate the precision of measurements, the linearity of the method, limit of quantification and detection (LOQ and LOD), specificity, accuracy, as well as metabolite stability. Values for the intra-day assays ranged from 0.2 to 3.2% while values for the inter-day assays ranged from 1.9 to 5.6% confirming that the method has good precision. A calibration plot using SA detected by GC/MS gave excellent linearity with a correlation coefficient of 0.999 over the injected concentration range of 5-2000 nmol/L. LOQ and LOD were 3 and 1 nmol/L, respectively. The usefulness of this method was demonstrated by SA quantification in an AF sample of an affected fetus and in plasma of patients treated with NTBC. The results demonstrate that this novel GC/MS method may be a valuable tool for metabolic evaluation and clinical use.     

Development of a novel LC-MS/MS method for the determination of letosteine in human plasma and its application on pharmacokinetic studies

Letosteine has been found to be effective in treating patients with chronic bronchopneumopathies in clinical practice. To provide robust support for its pharmacokinetic and clinical studies, a rapid and sensitive method based on liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) was developed and validated for the analysis of letosteine in plasma samples. After protein precipitation, the plasma samples were separated on a reversed-phase C(18) column in less than 1.5 min. The LC-MS/MS system was performed in the positive ion multiple-reaction-monitoring (MRM) mode to produce intensive product ions of m/z 280.1→160.0 for letosteine and m/z 248.1→121.1 for the internal standard, tinidazole. The method was found to have excellent linearity (r ≥ 0.9974), precision (RSD ≤ 5.83%), extraction recovery (71.8-73.0%) and stability (RE, -8.45% to 9.03%) over a concentration range of 0.1140-152.0 μgL(-1). Compared to the previous published radioactive method, LC-MS/MS method showed many advantages including shorter analysis time, simpler preparation procedure, increased sensitivity as well as lower safety risks. In addition, this method was successfully applied to study the pharmacokinetics of letosteine following a single and multiple dose oral administration in Chinese healthy volunteers.     

Determination of HS270, a new histone deacetylase inhibitor, in rat plasma by LC-MS/MS--application to a preclinical pharmacokinetic study

A rapid and sensitive liquid chromatography/tandem mass spectrometry (LC-MS/MS) method has been developed and fully validated to determine HS270, a new histone deacetylase (HDAC) inhibitor, in rat plasma using SAHA as the internal standard (IS). After a single step liquid-liquid extraction with acetoacetate, analytes were subjected to LC-MS/MS analysis using positive electro-spray ionization (ESI(+)) under selected reaction monitoring mode (SRM). The chromatographic separation was achieved on a Hypurity C(18) column (50 mm × 2.1 mm, i.d., 5 μm). The MS/MS detection was conducted by monitoring the fragmentation of m/z 392.3→100.1 for HS270, m/z 265.1→232.1 for IS. The method had a chromatographic running time of 2.5 min and linear calibration curves over the concentrations of 0.5-1000 ng/mL. The recovery of the method was 70.8-82.5% and the lower limit of quanti?cation (LLOQ) was 0.5 ng/mL. The intra- and inter-batch precisions were less than 15% for all quality control samples at concentrations of 1.0, 100.0, and 750.0 ng/mL. The validated LC-MS/MS method has successfully applied to a HS270 pharmacokinetic study after oral doses of 25, 50, 100, 200 mg/kg, and i.v. dose of 5 mg/kg to rats.     

Methylmalonic acid quantification in low serum volumes by UPLC-MS/MS

Methylmalonic acid (MMA) is a metabolic intermediate transformed to succinic acid (SA) by a vitamin B(12)-dependent catalytic step, and is broadly used as a clinical biomarker of functional vitamin B12 status. However, reported methods use between 100 and 1000 μL of serum or plasma making them sub-optimal for sample-limited studies, including those with neonates and infants. LC-MS/MS based protocols to measure MMA as n-butyl esters in the presence of tri-deuterated MMA (MMA-d(3)) were modified for use with 25 μL of human serum by scaling down sample processing volumes and analysis by UPLC-MS/MS. Plasma-based calibration solutions were found to be unnecessary, and chromatographic resolution and peak shape of SA and MMA was optimized in <4 min with isocratic 53:47 methanol/1.67 mM (pH 6.5) ammonium formate. Additionally, 1-cyclohexyl-urido-3-dodecanoic acid (CUDA) was included as internal standard allowing direct assessment of MMA recovery. Sample concentrations in the low normal range produced a signal:noise of >100:1. MMA intra- and inter-assay variability was under 10%. MMA-d(3) surrogate recovery averaged 93±14%. MMA stability exceeded three years in frozen samples and was unaffected by up to five freeze/thaw cycles. In conclusion, we report that methylmalonic acid can be measured with 25 μL of serum using water based standards. The assay signal:noise per concentration indicates that the method could perform as implemented with as little as 5 μL of serum. The reported method is applicable for studies of functional B12 status in sample limited experiments including investigations of nutritional status in neonates and in studies where low normal MMA levels are expected.     

A confirmatory analysis of malachite green residues in rainbow trout with liquid chromatography-electrospray tandem mass spectrometry

A quantitative liquid chromatography-tandem mass spectrometric (LC-MS/MS) method has been developed for the determination of malachite green (MG) and its metabolite leucomalachite green (LMG) in fish. Residues were extracted with an acetonitrile-acetate buffer and purified using the automated solid-phase extraction (ASPEC). Residues were analyzed with a reversed-phase LC-MS/MS using a positive-ion electrospray ionisation (ESI). Isotope-labelled leucomalachite green (LMG-D5) was used as an internal standard for the quantification of LMG residues. The related dye, brilliant green (BG) was used as an instrumental standard. Identification and quantification of analytes were based on the ion transitions monitored by multiple reaction monitoring (MRM). The decision limit (CCalpha) for MG and LMG was 0.13 and 0.16 microgkg(-1). The respective detection capabilities (CCbeta) were 0.22 and 0.27 microgkg(-1). The absolute recovery (repeatability SD(r)) was in the range of 58-65% (7.8-11.2%) for MG and 59-68% (9.7-16.9%) for LMG. LMG was quantified also based on the internal standard, giving a recovery (repeatability SD(r)) of 103-110% (4.8-9.3%). The method was further evaluated by analyzing a total of 34 fish residue monitoring samples, of which eight samples were found to be non-compliant containing low residues of LMG.     

Determination of picropodophyllin and its isomer podophyllotoxin in human serum samples with electrospray ionization of hexylamine adducts by liquid chromatography-tandem mass spectrometry

A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for determination of the new anticancer agent picropodophyllin (AXL1717) and its isomer podophyllotoxin levels in human serum has been developed. Monitoring of hexylamine adducts rather than proton adducts was used to optimize sensitivity. The chromatography system was an Acquity BEH C18, 2.1 mm × 50 mm 1.7 μm column with gradient elution (mobile phase A: 2.5 mM hexylamine and 5 mM formic acid in Milli-Q water and mobile phase B: methanol). The retention times were 1.4 min for picropodophyllin, 1.5 min for podophyllotoxin and 1.9 min for internal standard deoxypodophyllotoxin. The isomers were base-line separated. The analytes were detected after electrospray ionization in positive mode with selected reaction monitoring (SRM) with ion transitions m/z 516→102 for picropodophyllin and podophyllotoxin and m/z 500→102 for internal standard. The sample preparation was protein precipitation with acetonitrile (1:3) containing internal standard followed by dilution of the supernatant with mobile phase A (1:1). The limit of quantification (LOQ) was 0.01 μmol/L for picropodophyllin and podophyllotoxin. The limit of detection (LOD) at 3 times the signal to noise (S/N) was estimated below 0.001 μmol/L for picropodophyllin and podophyllotoxin. The quantification range of the method was between 0.01 μmol/L and 5 μmol/L for both isomers. The accuracy was within ±15% of the theoretical value for both picropodophyllin and podophyllotoxin and inter-assay precision did not exceed ±15%, except for the 0.016 μmol/L level of podophyllotoxin, which was 18%. The selectivity of the method was verified by analysis of two different product ions for each analyte and by analysis for interference of seven different batches of blank human serum. The combined recovery and matrix effects were about 83% for picropodophyllin and podophyllotoxin. The new LC-MS/MS method showed sufficient sensitivity and selectivity for determination of picropodophyllin and its isomer podophyllotoxin levels in human serum from subjects receiving therapeutic doses of AXL1717.     

Development and validation of a LC-MS/MS method for the determination of viaminate in human plasma

A sensitive and specific method for determination of viaminate in human plasma by using high-performance liquid chromatography coupled with electrospray tandem mass spectrometry (LC-MS/MS) was developed in this study. The plasma samples were simply deproteinated, extracted, evaporated, and then reconstituted in 200 microl of methanol prior to analysis. Chromatographic separation was carried out on a Shimadzu VP-ODS column (250 mm x 2.0 mm, 5 microm) with a mobile phase of methanol-water (95:5, v/v) at a flow rate of 0.2 ml/min. Quantification was performed in the negative-ion electrospray ionization mode by selected ion monitoring of the product ions at m/z 164 for viaminate and m/z 109 for testosterone propionate which was used as the internal standard. The corresponding parent ions were m/z 446 and m/z 345. A linear calibration curve was observed within the concentration range of 0.10-200 ng/ml. The lowest limit of quantitation (LLOQ) was 0.1 ng/ml. The extraction-efficiency at three concentrations was 100.7, 93.6, and 99.7%. Practical utility of this new LC-MS/MS method was confirmed in pilot pharmacokinetic studies in humans following oral administration.     

Development and validation of a liquid chromatography/tandem mass spectrometry assay for the simultaneous determination of nateglinide, cilostazol and its active metabolite 3,4-dehydro-cilostazol in Wistar rat plasma and its application to pharmacokinetic study

Nateglinide (NTG), an insulin secretogogue, has been studied in rats for drug-drug interaction with cilostazol (CLZ), an antiplatelet agent commonly used in diabetics. We developed a liquid chromatography tandem mass spectrometry (LC-MS/MS) based method that is capable of simultaneous monitoring plasma levels of nateglinide, cilostazol, and its active metabolite 3,4-dehydro-cilostazol (DCLZ). All analytes including the internal standard (Repaglinide) were chromatographed on reverse phase C(18) column (50 mm x 4.6mm i.d., 5 microm) using acetonitrile: 2mM ammonium acetate buffer, pH 3.4 (90:10, v/v) as mobile phase at a flow rate 0.4 ml/min in an isocratic mode. The detection of analyte was performed on LC-MS/MS system in the multiple reaction monitoring (MRM) mode. The quantitations for analytes were based on relative concentration. The method was validated over the concentration range of 20-2000 ng/ml and the lower limit of quantitation was 20 ng/ml. The recoveries from spiked control samples were >79% for all analytes and internal standard. Intra- and inter-day accuracy and precision of validated method were with in the acceptable limits of <15% at all concentration. The quantitation method was successfully applied for simultaneous estimation of NTG, CLZ and DCLZ in a pharmacokinetic drug-drug interaction study in Wistar rats.     

Quantification of 3-deazaneplanocin A, a novel epigenetic anticancer agent, in rat biosamples by hydrophilic interaction liquid chromatography-tandem mass spectrometric detection

A sensitive and selective LC-MS/MS based bioanalytical method was developed and validated for the quantification of 3-deazaneplanocin A (DZNep), a novel epigenetic anti-tumor drug candidate, in Sprague-Dawley (SD) rat biosamples (plasma, urine, feces and tissue samples). The method comprises a phenylboronic acid (PBA)-containing solid phase extraction procedure, serving for binding and clean-up of DZNep in rat biosamples spiked with tubercidin (as internal standard). The analytes were separated on an Agilent hydrophilic interaction chromatography (HILIC) column. LC-MS/MS in positive ion mode was used to perform multiple reaction monitoring at m/z of 263/135 and 267/135 for DZNep and tubercidin, respectively. The limit of quantification (LOQ) of DZNep in rat biosamples was 20 ng/mL. The data of intra-day and inter-day accuracy were within 15% of nominal concentration while the precision (relative standard deviation) less than 10% for all biosamples. The extraction recoveries for DZNep and tubercidin were consistent and reproducible (around 80%) and the matrix effects were negligible (around 10% suppression) in all biosamples. This method was demonstrated to be applicable for pharmacokinetic studies of DZNep in SD rats.     

Analysis of glucosinolates, isothiocyanates, and amine degradation products in vegetable extracts and blood plasma by LC-MS/MS

Dietary glucosinolates are under intensive investigation as precursors of cancer-preventive isothiocyanates. Quantitation of the dose and bioavailability of glucosinolates and isothiocyanates requires a comprehensive analysis of the major dietary glucosinolates, isothiocyanates, and related metabolites. We report a liquid chromatography with tandem mass spectrometric detection (LC-MS/MS) analytical method for the comprehensive analysis of the seven major dietary glucosinolates, related isothiocyanates, and putative amine degradation products. The parent glucosinolates were sinigrin, gluconapin, progoitrin, glucoiberin, glucoraphanin, glucoalyssin, and gluconasturtiin. The LC-MS/MS analysis method for these compounds was developed and validated; a standard addition analysis protocol was used generally to avoid the requirement for stable isotopic standards. Where stable isotopic standards were available, internal standardization with these gave estimates in agreement with those obtained by the standard addition analysis protocol. For glucosinolates, negative ion electrospray LC-MS/MS analysis was performed. Isothiocyanates and amines were prederivatized to the corresponding thiourea and N-acetamides, respectively, and were quantified by positive ion electrospray LC-MS/MS. The limits of detection were 0.5-2 pmol; the recoveries for glucosinolates, isothiocyanates, and amines were 85-90%, 50-85%, and 60-70%, respectively; and the intra- and interbatch coefficients of variation were 1-4% and 3-10%, respectively. These methods provide facile access to comprehensive analytical data on the major dietary glucosinolates and related metabolites to quantify inputs and metabolic formation of these compounds in cancer prevention and related studies.     

Simultaneous quantification of the new HIV protease inhibitors atazanavir and tipranavir in human plasma by high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry

We have developed and validated an assay, using liquid chromatography coupled with electrospray tandem mass spectrometry (LC-MS/MS), for the quantification of the novel protease inhibitors (PIs) atazanavir and tipranavir. The sample pre-treatment consisted of protein precipitation with a mixture of methanol and acetronitrile using 100 microl plasma for atazanavir and 50 microl for tipranavir. Chromatographic separation was achieved on an Inertsil ODS3 column (50 mm x 2.0 mm i.d., particle size 5 microm), with a quick stepwise gradient using an acetate buffer (pH 5) and methanol, at a flow rate of 0.5 ml/min. The analytical run time was 5.5 min. The triple quadrupole mass spectrometer operated in the positive ion-mode and multiple reaction monitoring (MRM) was used for drug quantification. The assay was linear over a concentration range of 0.05-10 microg/ml for atazanavir and 0.1-75 microg/ml for tipranavir. Saquinavir-d5 was used as internal standard. The intra- and inter-day coefficients of variation were less than 3.8% for atazanavir and less than 10.4% for tipranavir. Accuracies were within +/-7.3 and +/-7.2% for atazanavir and tipranavir, respectively. Both drugs were stable under various relevant storage conditions. The validated concentration ranges proved to be adequate to measure concentrations of human immunodeficiency virus type-1 (HIV-1)-infected individuals. The developed method could easily be combined with a previously developed LC-MS/MS assay for the quantification of protease inhibitors.     

Determination of isometamidium residues in animal-derived foods by liquid chromatography-tandem mass spectrometry

In this paper, a new determination method for isometamidium residues in animal-derived foods was developed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Isometamidium residues in bovine tissues and milk were extracted with the mixed solution of acetonitrile and 0.25 mol/L of ammonium formate-methanol (v/v, 1:1), concentrated and degreased, and determined by LC-MS/MS with quantification by external standard method. The results showed that the peak area of chromatogram was linearly related to the concentration of isometamidium in the range of 1-100 μg/L, and the limits of detection (LOD) and quantification (LOQ) were 0.05 μg/kg and 5 μg/kg, respectively. The average recoveries of spiked samples were in the range of 73.8-93.9% with relative standard deviations ranged from 2.3% to 7.5%. This method is simple, accurate and suitable for the identification and quantification for isometamidium in animal-derived foods.     

Clinical determination of 17-hydroxyprogesterone in serum by LC-MS/MS: comparison to Coat-A-Count RIA method

17alpha-Hydroxyprogesterone is a metabolic precursor of cortisol; elevated levels of 17alpha-hydroxyprogesterone are indicative of congenital adrenal hyperplasia. Traditional determination by immunoassay is plagued by poor antibody specificity, resulting in significant interferences. This study explores an LC-MS/MS method for the quantitation of 17OHP in serum. Deuterated 17alpha-hydroxyprogesterone was added as internal standard, followed by solid-phase extraction, HPLC separation with a C16-amide reverse-phase column with run time of 7 min, and quantification by MS/MS (positive electrospray ionisation) in the selected reaction monitoring mode (SRM). Transitions monitored were 331>109 for the analyte and 339>113 for the deuterated internal standard. Intra-assay precision (%R.S.D.) was 7.4% at 7 nmol/L, inter-assay precision (%R.S.D.) at 2, 7 and 27 nmol/L was 15.4, 10.0 and 7.9% and accuracy at 0.9 nmol/L was 100%. The method was linear from 0.156 to 80 nmol/L. Lower limit of quantitation was 0.2 nmol/L, providing meaningful data for patients within normal range as well as those with elevated levels.     

Quantitation of desmosine and isodesmosine in urine, plasma, and sputum by LC-MS/MS as biomarkers for elastin degradation

The aim of this study is to develop a standardized LC-MS/MS method for accurate measurement of desmosine (DES) and isodesmosine (IDS) in all body fluids as biomarkers for in vivo degradation of matrix tissue elastin in man and animals. A reproducible three-step analytical procedure: (1) sample hydrolysis in 6N HCl, (2) SPE by a CF1 cartridge with addition of acetylated pyridinoline as internal standard (IS), and (3) LC/MSMS analysis by SRM monitoring of transition ions; DES or IDS (m/z 526-481+397) and IS (m/z 471-128) was developed. The method achieves accurate measurements of DES/IDS in accessible body fluids (i.e. urine, plasma, and sputum). LOQ of DES/IDS in body fluids is 0.1 ng/ml. The % recoveries and reproducibility from urine, plasma, and sputum samples are above 99 ± 8% (n = 3), 94 ± 9% (n = 3) and 87 ± 11% (n = 3), with imprecision 8%, 9% and 10%, respectively. The proposed method was applied to measure DES/IDS in body fluids of patients with chronic obstructive pulmonary disease (COPD) and healthy controls. Total DES/IDS in sputum and plasma is increased over normal controls along with the free DES/IDS in urine in patients. DES/IDS can be used to study the course of COPD and the response to therapy. This practical and reliable LC-MS/MS method is proposed as a standardized method to measure DES and IDS in body fluids. This method can have wide application for investigating diseases which involve elastic tissue degradation.     

Liquid chromatography/electrospray ionisation-mass spectrometry method for the quantification of sphingosine and sphinganine in cell cultures exposed to fumonisins

Fumonisins, mycotoxins produced by Fusarium verticillioides, are potent inhibitors of the de novo sphingolipid biosynthesis via inhibition of the key enzyme ceramide synthase. The cellular response to a fumonisin exposure is obvious as an alteration of the ratio of the sphingoid bases sphingosine (SO) and sphinganine (SA). We developed a new column liquid chromatography/electrospray ionisation-mass spectrometry (LC-ESI-MS) method for the rapid, simultaneous and quantitative determination of these bases in cell cultures of immortalised human kidney epithelial cells (IHKE cells). For sample preparation, cell lysates were only diluted, centrifuged and directly used for LC-MS measurements. Quantification was carried out using phytosphingosine (PSO) as an internal standard. Detecting the protonated molecule [M+H](+) signals of SO (m/z 300) and SA (m/z 302) in the selected ion monitoring (SIM) mode, detection limits of 10 pg for SO (signal-to-noise ratio S/N=3:1) and 25 pg for SA (S/N=3:1) were established. The average recovery for SO and SA was higher than 90% for control IHKE-cells, respectively. The developed LC-ESI-MS method allows the sensitive, selective and rapid monitoring of sphingosine and sphinganine in cell matrices with a drastically reduced time for sample preparation.     

The determination of gemcitabine and 2'-deoxycytidine in human plasma and tissue by APCI tandem mass spectrometry

A fast, sensitive and accurate method for the determination of gemcitabine (difluorodeoxycytidine; dFdC) and deoxycytidine (CdR) in human plasma/tissue was developed using LC-MS/MS techniques. Effectiveness of the method is illustrated with the analysis of plasma from a phase I trial of dFdC administered as a 24h infusion. The method was developed using (15)N(3) CdR as an internal standard across the concentration range of 1-500ng/ml, using a cold alcohol-protein precipitation followed by desorption with freeze drying. Sample clean-up for LC-MS/MS analysis was performed by an innovative liquid/liquid back extraction with ethyl acetate and water. Chromatography was performed using a Chrompak-spherisorb-phenyl-column (3.1mmx200mm, 5microm) with a 50mM formic acid: acetonitrile (9:1) mobile phase eluted at 1ml/min. Extracted samples were observed to be stable for a minimum of 48h after extraction when kept at 4 degrees C. Detection was performed using an atmospheric pressure chemical ionization (APCI) source and mass spectrometric positive multi-reaction-monitoring-mode (+MRM) for dFdC (264 m/z; 112 m/z), CdR (228 m/z; 112 m/z), and (15)N(3) CdR (231 m/z; 115 m/z) at an ion voltage of +3500V. The accuracy, precision and limit-of-quantitation (LOQ) were as follows: dFdC: 99.8%, +/-7.9%, 19nM; CdR: 100.0%, +/-5.3%, 22nM, linear range LOQ to 2microM. During 24h infusion dFdC levels were detected with no interference from either CdR or difluorodeoxyuridine (dFdU). CdR co-eluted with dFdC but selectivity demonstrated no "crosstalk" between the compounds. In conclusion the analytical assay was very sensitive, reliable and robust for the determination of plasma and tissue concentrations of dFdC and CdR.