Urine profiling by SELDI-TOF/MS: monitoring of the critical steps in sample collection, handling and analysis

The topic of this study is the impact of several pre-analytical and analytical variables on proteomic profiling of human urine by surface enhanced laser desorption/ionization time of flight-mass spectrometry (SELDI-TOF-MS) in healthy subjects. Urine storage at room temperature caused a progressive degradation of proteins, which was prevented by the addition of protease inhibitors only up to 2 h from the collection. The timing of collection over the day had only a minor impact on protein profile, although influencing the intensity of peaks. Repeated freeze/thaw cycles (up to five) did not affect either the number or the intensity of the peaks. A comparison of the protein profile from eight different healthy individuals showed fairly consistent inter-subject similarities, along with between-subject differences, which were markedly dependent on the sex and the type of ProteinChip array used. The addition of a variety of denaturing agents improved the quality of the spectra with all the chips tested (CM10, Q10 and H50), but not with the copper-coated IMAC-30 chip. Finally, SPA matrix allowed to achieve a better performance of SELDI-TOF/MS spectrum, as compared with CHCA, regardless of the ProteinChip array used and even in the low m/z range (2500-10,000). In conclusion, we suggest that a careful choice of a number of pre-analytical and analytical conditions is required to accomplish and define a unifying protocol for the analysis of human urine by SELDI-TOF/MS, in physiological and in pathological states.     

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.     

Usefulness of multi-parameter opiates-amphetamines-cocainics analysis in hair of drug users for the evaluation of an abuse profile by means of LC-APCI-MS-MS

The report presents a segmental hair analysis for the retrospective multi-parameter evaluation of drugs of abuse including opioids, cocainics and amphetamines. The analysis was carried out with the use of liquid chromatography atmospheric pressure chemical ionization tandem mass spectrometry (LC-APCI-MS-MS). The authors have evaluated the differences in the contents of particular opiates in the hair as related to the origin of a sample taken from Polish drug users taking "Polish heroin", and also from heroin abusers from Western European countries taking "Western heroin". The results indicate distinct differences in the 6-MAM concentration values in the Polish and foreigners, suggesting that the foreigners take products containing high concentrations of heroin and the Polish take the poppy product "compote" characterized by its variable and low heroin content. An additional argument for a different abuse profile in the Polish and Western drug users is found in the presence of cocaine detected in hair samples originating from the latter, while cocaine is much less frequently detected in Polish drug users.     

Validated HPLC method for the determination of gabapentin in human plasma using pre-column derivatization with 1-fluoro-2,4-dinitrobenzene and its application to a pharmacokinetic study

A rapid, sensitive and accurate high-performance liquid chromatographic method with UV detection was developed and validated for the quantification of gabapentin in human plasma. Gabapentin was quantified using pre-column derivatization with 1-fluoro-2,4-dinitrobenzene following protein precipitation of plasma with acetonitrile. Amlodipine was used as internal standard. The chromatographic separation was carried out on a Nova-Pak C(18) column using a mixture of 50 mM NaH(2)PO(4) (pH=2.5)-acetonitrile (30:70, v/v) as mobile phase with UV detection at 360 nm. The flow rate was set at 1.5 ml/min. The method was linear over the range of 0.05-5 microg/ml of gabapentin in plasma (r(2)>0.999). The within-day and between-day precision values were in the range of 2-5%. The limit of quantification of the method was 0.05 microg/ml. The method was successfully used to study the pharmacokinetics of gabapentin in healthy volunteers.     

Simultaneous determination of 6 beta-blockers, 3 calcium-channel antagonists, 4 angiotensin-II antagonists and 1 antiarrhythmic drug in post-mortem whole blood by automated solid phase extraction and liquid chromatography mass spectrometry. Method development and robustness testing by experimental design

A method for the simultaneous determination of the beta-blockers atenolol, sotalol, metoprolol, bisoprolol, propranolol and carvedilol, the calcium-channel antagonists diltiazem, amlodipine and verapamil, the angiotensin-II antagonists losartan, irbesartan, valsartan and telmisartan, and the antiarrhythmic drug flecainide, in whole blood samples from forensic autopsies was developed. Sample clean-up was achieved by precipitation and solid phase extraction (SPE) with a mixed-mode column. Quantification was performed by reversed phase high performance liquid chromatography with positive electrospray ionization mass spectrometric detection (HPLC-MS). The method has been developed and robustness tested by systematically searching for satisfactory conditions using experimental designs including factorial and response surface designs. With the exception of amlodipine, the concentration limit of quantification (cLOQ) covered low therapeutic concentration levels for all the compounds. Within assay precisions and accuracies (bias) were 3.4-21% RSD and from -24 to 21% for the concentration range 1.00-5.00 microM, respectively. Between assay precisions were 4.4-28% RSD for the concentration range from 0.1 to 5 microM and recoveries varied from 9 to 103%. The method is used for determination of cardiovascular drugs in post-mortem whole blood samples from forensic autopsy cases.     

Sensitive determination of aspirin and its metabolites in plasma by LC-UV using on-line solid-phase extraction with methylcellulose-immobilized anion-exchange restricted access media

We describe a sensitive determination of aspirin (ASA) and its three metabolites (salicylic acid [SA], 2,3-dihydroxybenzoic acid [2,3-DHBA], and 2,5-dihydroxybenzoic acid [gentisic acid (GA)]) in rat plasma. Analysis was carried out by on-line solid-phase extraction (SPE) using a methylcellulose-immobilized-strong anion-exchanger (MC-SAX), followed by liquid chromatography (LC) coupled with UV detection. The lower limits of quantitation for ASA and SA were 60 ng/mL in 100 microL of plasma, respectively. This method was validated with respect to intra- and inter-day precision, accuracy, and linearity up to concentrations of 20,000 ng/mL for ASA, SA, 2,3-DHBA and gentisic acid, respectively. The method was successfully applied to an analysis of the pharmacokinetics of ASA and SA in rats.     

Simple and rapid method determination for metformin in human plasma using high performance liquid chromatography tandem mass spectrometry: application to pharmacokinetic studies

A rapid and simple method for quantitation of metformin (MET) in human plasma by HPLC-MS/MS was developed and validated. The sample preparation consists of plasma deproteinization using acetonitrile. The mobile phase consisted of water-acetonitrile and formic acid (55/45/0.048, v/v/%) and the run time was 3 min. A pursuit C(18) (100 mm x 2.0 mm i.d., 3 microm) column connected to a guard column MS-pursuit (0.20 mm x 0.20 mm i.d., 5 microm) was used. The range of the calibration curve was from 20 to 5000 ng/mL, the limit of quantitation being 20 ng/mL. The detection was performed on a mass spectrometer (ESI+), using metoprolol as internal standard. The calibration curves have r(2) values of 0.995 (CV=0.24%, n=10). The accuracy and precision were between 90.74 and 106.7% and coefficients of variations (CV) of 1.10 and 4.35%, respectively. The method was applied to determine the pharmacokinetic parameters: C(max) (1667.25 ng/mL) and T(max) (3.89 h).     

Validation of a rapid micellar electrokinetic capillary chromatographic method for the simultaneous determination of isoniazid and pyridoxine hydrochloride in pharmaceutical formulation

An efficient and reliable micellar electrokinetic capillary chromatography (MEKC) method has been developed for the simultaneous determination of isoniazid (ISO) and pyridoxine hydrochloride (PYR) in pharmaceutical formulations. A chemometric two level full factorial design approach was used to search for the optimum conditions of separation. Three parameters were selected for this study: the buffer pH, the buffer concentration and sodium dodecyl sulphate (SDS) concentrations. Resolution, peak symmetry and analysis time were established as response. The two analytes were separated within 6 min with the optimized conditions: 50 mM borate buffer, 25 mM SDS pH 7.8, 35 degrees C, at 50 mbar 4s injection and 30 kV by using a fused silica capillary (72 cm effective length, 50 microm i.d.). The detection wavelength was set to 205 nm. Meloxicam was used as internal standard. The method was validated with respect to stability, linearity range, limit of quantitation and detection, precision, accuracy, specificity and robustness. The detection limits of the method were 1.0 microg mL(-1) for ISO and 0.40 microg mL(-1) for PYR and the method was linear at least in the range of 3.0-100 microg mL(-1) for ISO and 1.0-100 microg mL(-1) for PYR with excellent correlation coefficients (0.9995 for ISO and 0.9998 for PYR). Relative standard deviations (R.S.D.s) of the described method ranged between 0.54 and 2.27% for intra-day precision and between 0.65 and 2.69% for inter-day precision. The developed method was applied to the tablet form of ISO and PYR-containing the pharmaceutical preparations and the data were compared with obtained from the standard addition method. No statistically significant difference was found.     

Quantitative determination of the macrolide antibiotic tulathromycin in plasma and broncho-alveolar cells of foals using tandem mass spectrometry

The long-acting antibiotic tulathromycin is on the marked for treatment of pulmonary infection of cattle, swine and horses. To measure disposition and distribution of tulathromycin in foals, a high throughput method was developed for horse plasma (calibration range: 0.006-0.8 microg/mL) and broncho-alveolar cells (calibration range: 0.1-4.0 microg/10(9)cells) using tandem mass spectrometry. Tulathromycin was extracted from plasma and broncho-alveolar fluid using cation exchange cartridges with acetonitrile/ammonia (95:5, v/v). The chromatography was performed isocratically with a mobile phase consisting of 5 mM ammonium formiate buffer/acetonitrile (30:70, v/v). The mass spectrometer operated in selected ion mode with atmospheric pressure chemical ionization to monitor the respective MH+ ions m/z 577.3 for tulathromycin and m/z 679.3 for the internal standard roxithromycin. All quality parameters fulfilled the international criteria for bioanalytical method validation and were successfully applied to the determination of tulathromycin in plasma of foals and broncho-alveolar cells. In foals, tulathromycin after intramuscular administration was rapidly absorbed, widely distributed and slowly eliminated. It cumulated manifold in broncho-alveolar cells.     

Systematic and comprehensive strategy for reducing matrix effects in LC/MS/MS analyses

A systematic, comprehensive strategy that optimizes sample preparation and chromatography to minimize matrix effects in bioanalytical LC/MS/MS assays was developed. Comparisons were made among several sample preparation methods, including protein precipitation (PPT), liquid-liquid extraction (LLE), pure cation exchange solid-phase extraction (SPE), reversed-phase SPE and mixed-mode SPE. The influence of mobile phase pH and gradient duration on the selectivity and sensitivity for both matrix components and basic analytes was investigated. Matrix effects and overall sensitivity and resolution between UPLC technology and HPLC were compared. The amount of specific matrix components, or class of matrix components, was measured in the sample preparation extracts by LC/MS/MS with electrospray ionization (ESI) using both precursor ion scanning mode and multiple reaction monitoring (MRM). PPT is the least effective sample preparation technique, often resulting in significant matrix effects due to the presence of many residual matrix components. Reversed-phase and pure cation exchange SPE methods resulted in cleaner extracts and reduced matrix effects compared to PPT. The cleanest extracts, however, were produced with polymeric mixed-mode SPE (both reversed-phase and ion exchange retention mechanisms). These mixed-mode sorbents dramatically reduced the levels of residual matrix components from biological samples, leading to significant reduction in matrix effects. LLE also provided clean final extracts. However, analyte recovery, particularly for polar analytes, was very low. Mobile phase pH was manipulated to alter the retention of basic compounds relative to phospholipids, whose retention tends to be relatively independent of pH. In addition to the expected resolution, speed and sensitivity benefits of UPLC technology, a paired t-test demonstrated a statistically significant improvement with respect to matrix effects when this technology was chosen over traditional HPLC. The combination of polymeric mixed-mode SPE, the appropriate mobile phase pH and UPLC technology provides significant advantages for reducing matrix effects resulting from plasma matrix components and in improving the ruggedness and sensitivity of bioanalytical methods.