Determination of malachite green and leucomalachite green in edible goldfish muscle by liquid chromatography-ion trap mass spectrometry

A liquid chromatography-ion trap mass spectrometry method with three "time segments" has been developed to determine malachite green (MG) and its major metabolite, leucomalachite green (LMG) in edible goldfish muscle. By using the optimized "time segments", MG and LMG as well as the internal standard atrazine-d(5) were analyzed with good sensitivity with positive ESI-MS in a single run. The homogenized fish muscle tissues were extracted with a solution of perchloric acid and acetonitrile, followed by partitioning with dichloromethane. Strata-x polymeric solid-phase extraction column was used for the clean-up process. The determination of MG and LMG was achieved by using a reversed-phase HPLC gradient program coupled with MS/MS in multiple-reaction-monitoring mode. Matrix calibration curves were linear over the ranges of 5-500 ng/ml for MG and 1-100 ng/ml for LMG. Recoveries of the fish tissue extraction at three spiked levels (2, 10 and 30 ng/g for MG as well as 0.4, 2 and 6 ng/g for LMG) were better than 71% and 89%, respectively. Relative standard derivations from six determinations were less than 8%. The method detection limits were 0.13 ng/g for MG and 0.06 ng/g for LMG.     

Identification of incurred sulfonamide residues in eggs: methods for confirmation by liquid chromatography-tandem mass spectrometry and quantitation by liquid chromatography with ultraviolet detection

Two complementary methods for identifying and measuring sulfonamide residues in eggs were developed for use in surveying eggs for potential drug residues. The first method uses liquid chromatography-tandem mass spectrometry (LC-MS-MS) to confirm the presence of sulfonamide residues in eggs. During its validation the limit of confirmation was estimated to be 5-10 ng/g (ppb) depending on the drug. Also, a method for measuring residue level by liquid chromatography with ultraviolet detection (LC-UV) was validated using the same extraction procedure as the confirmatory method. The determinative method was validated over the 50-200 ppb range. Samples were prepared by homogenizing whole egg, extracting with acetonitrile, and cleaning up with a C(18) solid-phase extraction cartridge. For confirmation, analytes were separated by gradient LC on a C(18) column, ionized by electrospray ionization (ESI), and detected by MS-MS with an ion trap mass spectrometer. For determination, analytes were separated by a different gradient LC procedure and detected by UV at 287 nm. Fifteen drugs were dosed individually in laying hens, and residues of parent drug and/or metabolites were found in eggs for all the drugs. Validation was based on repetitive analyses of control samples, control samples fortified at 100 ppb sulfonamides, and samples of blended incurred eggs.     

Simultaneous determination of malachite green, gentian violet and their leuco metabolites in catfish or trout tissue by high-performance liquid chromatography with visible detection

A sensitive analytical procedure for the determination of residues of leucomalachite green (LMG)-malachite green (MG) and leucogentian violet (LGV)-gentian violet (GV) in catfish or trout tissue is presented. Frozen (−20°C) fish fillets were cut into small pieces and blended in a Waring blender. A 20-g amount of homogenized fish tissue was extracted with acetonitrile-buffer, partitioned against methylene chloride, and cleaned up on tandem neutral alumina and propylsulfonic acid cation-exchange solid-phase extraction cartridges. Samples of 100 μl (0.8 g equiv.) were chromatographed isocratically in 10 min using an acetonitrile-buffer mobile phase on a short-chain deactivated (SCD) reversed-phase column (250×4.6 mm I.D.) in-line with a post-column PbO₂ oxidation reactor. The PbO₂ post-column reactor efficiently oxidized LMG to the chromatic MG, and LGV to the chromatic GV permitting visible detection at 588 nm for all four compounds. Linearity was demonstrated with standards over the range of 0.5–50 ng per injection. Recoveries of LMG, MG, LGV and GV from catfish tissues fortified at 10 ng/g were 75.4±3.0, 61.3±4.1, 72.6±3.7 and 87.9±2.5, respectively, while trout tissues fortified at 10 ng/g yielded recoveries of 82.6±2.3, 48.6±1.8, 72.1±2.1 and 83.8±4.6 (mean±S.D., N=4), respectively.     

High accuracy determination of malachite green and leucomalachite green in salmon tissue by exact matching isotope dilution mass spectrometry

A high accuracy method for the quantification of malachite green (MG) and leucomalachite green (LMG) in salmon is described. Analytical challenges including the effects of analyte instability and matrix suppression were minimised by the use of exact matching isotope dilution mass spectrometry. The developed method included overnight extraction in acidified acetonitrile/ammonium acetate buffer and analysis by LC-MS/MS utilising isotopic internal standards. This method was used to determine the level of MG and LMG in a sample of salmon used in an international inter-comparison organised by the Comité Consultatif pour la Quantité de Matière (CCQM). The sum of MG and LMG was found to be 9.32+/-0.98ngg(-1) at the 95% confidence interval (relative expanded uncertainty 10.5% (k=2)). This encompassed the mean and median of the CCQM inter-comparison.     

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.     

Rapid identification of vinca alkaloids by direct-injection electrospray ionisation tandem mass spectrometry and confirmation by high-performance liquid chromatography-mass spectrometry

A simple and rapid method for the identification of Vinca alkaloids from a crude extract of Catharanthus roseus G. Don (Apocynaceae) by direct-injection electrospray ionisation (ESI) and tandem mass spectrometry (MS/MS) has been developed. The alkaloids vindoline, vindolidine, vincristine and vinblastine were evaluated in a commercial extract of C. roseus using this method. Catharanthine and its isomers 19S-vindolinine and vindolinine were detected in the commercial product by direct injection ESI/MS/MS and confirmed by preparation and by HPLC-ESI/MS. For the characterisation of different fragment fingerprints, ESI/MS/MS is a sensitive, rapid and convenient technique by which to identify some constituents in complex and mixed plant extracts.     

Determination of paclitaxel in mouse plasma and brain tissue by liquid chromatography-mass spectrometry

Paclitaxel is an anticancer agent extracted from the bark of the yew tree and is widely used in chemotherapy for solid tumors, including non-small cell lung cancer and ovarian carcinoma. Most assays to measure paclitaxel in plasma require a large amount of sample (0.4-1 ml) to achieve the necessary sensitivity, and are not suitable when only small sample sizes are available. To circumvent this latter limitation, we developed a sensitive liquid chromatography-mass spectrometry (LC-MS) method for the determination of paclitaxel in plasma based on the use of small sample volumes (50 microl plasma). A solid phase extraction procedure was employed that enabled the eluent to be directly injected onto a reversed phase chromatographic HPLC system using positive electrospray ionization followed by mass spectrometric detection. The extraction recoveries of paclitaxel were 98 and 83% from plasma and brain tissues, respectively. The mobile phase consisted of 50% acetonitrile in 0.1% formic acid that was pumped at 0.2 ml/min to yield a retention time for paclitaxel of 6.2 and 5.4 min for cephalomannine, the internal standard. The method has been validated at paclitaxel plasma concentrations from 0.036 to 9.9 microg/ml, and from 0.054 to 1.96 microg/ml in brain homogenates. A sensitive and specific assay for paclitaxel has been developed that has the advantages of using small sample sizes, and a single extraction step without solvent evaporation.     

Observations on malachite green in relation to its application to fish diseases

Summary Experiments were carried out to evaluate several properties determining the mutual influence of the system: fish-bath-malachite green-pathogen.The toxicity of malachite green solutions in concentrations varying between 1 : 80,000 and 1 : 2,560,000 was tested on immature sand whiting. As regards the toxicity of 1 : 80,000 solutions, for practical purposes no distinction between fish of different size, between equal concentrations of chloride and oxalate salts of the dye, or between fresh and decolourized solutions appears necessary.The survival time increased appreciably with decreasing temperature for the range 13.5–28.0°C.The dye solutions always showed significant decolourization when the pH was higher than 5.Several body tissues of the fish, notably the central nervous system and the lateral red muscles, were stained by fresh as well as decolourized malachite green solutions.The dye proved bacteriostatic but not bactericidal, to pseudomonad and coccus cultures when 1 : 80,000 solutions were used. Once decolourized, the dye became a less effective bacteriostatic.The addition of the dye, or formaldehyde, to the bath induced hyperventilation in the fish.A triple dye mixture, consisting of malachite green, brilliant green and crystal violet, was found to be a valuable therapeutic agent.

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Zusammenfassung Einige Versuche werden durchgeführt zur Erklärung verschiedener Eigenschaften, die die gegenseitige Einwirkung in dem System: Fisch - Bad - Malachitgrün - Pathogen bestimmen.Die Toxizität von Malachitgrün-Lösungen in Konzentrationen welche variierten zwischen 1 : 80,000 and 1 : 2,560,000 wurde geprüft an jungen Sand Whiting. Was die Toxizität der 1 : 80,000 Lösungen betrifft, scheint, für praktischen Zwecke, kein Unterschied zu sein zwischen Fischen verschiedener Länge, zwischen gleichen Konzentrationen von Chlorid und Oxalat-Salzen des Farbstoffes, oder zwischen frischen und entfärbten Lösungen. Die Überlebungszeit nahm merklich zu, wenn die Temperatur erniedrigt war, im Gebiet von 13.5°–28.0°C. Die Farbstoff-Lösungen zeigten erhebliche Entfärbung, wenn der pH - Wert grösser war als 5. Verschiedene Körpergewebe des Fisches, insbesondere das Zentralnervensystem und die lateralen roten Muskeln, werden von frischen sowie entfärbten Lösungen grün gefärbt. Der Farbstoff zeigte in 1 : 80,000 Lösungen bakteriostatische, aber keine bakterizide Wirkung, wenn geprüft an Pseudomonas und Kokkus Kulturen. Zusatz des Farbstoffes, oder Formaldehyde, zum Bade veranlasste in dem Fisch Hyperventilation. Ein Tripelfarbstoff-Gemisch, das bestand aus Malachitgrün, Brilliantgrün und Kristallviolett, zeigte sich ein wertvolles Therapeutikum.

     

Determination of cediranib in mouse plasma and brain tissue using high-performance liquid chromatography-mass spectrometry

A new high performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS) assay for cediranib, a tyrosine kinase inhibitor for VEGFRs, was developed and validated, for the determination of plasma and brain levels of cediranib in small specimen volumes. Tyrphostin (AG1478) was used as internal standard. Mouse plasma and brain homogenate samples were prepared using liquid-liquid extraction. The assay was validated for a 2.5-2500 ng/mL concentration range for plasma, and for 1-2000 ng/mL range for brain homogenate. For these calibration ranges, within-assay variabilities were 1.1-14.3% for plasma and 1.5-9.4% for brain homogenate; between-assay variabilities were 2.4-9.2% for plasma, and 4.9-10.2% for brain homogenate. Overall accuracy ranged from 101.5 to 107.0% for plasma and 96.5 to 100.2% for brain homogenate, for all target concentrations. The developed assay has been successfully applied for a brain distribution study in mice at an oral dose of 5 mg/kg.     

An improved malachite green assay of phosphate: mechanism and application

The classical malachite green (MLG) assay of phosphate, which added MLG after molybdate to the acidified reaction solutions of phosphate, tolerated interference from papaverine, sildenafil, and some similar hydrophobic amines. Resonance Rayleigh scattering signals, the alleviation of interference by poly(vinyl alcohol), and the precipitation of some yellow complexes supported that the irreversible aggregation of the complexes of a hydrophobic amine of interference and phosphomolybdate reduced the amounts of phosphomolybdate accessible to MLG and caused the interference. By adding MLG before molybdate to the acidified reaction solutions of phosphate, the complexes of phosphomolybdate and MLG were preferentially formed before the complexes of phosphomolybdate and such a hydrophobic amine effectively aggregated; thereby, an improved MLG assay of phosphate with the resistance to common hydrophobic amines was developed. Using the improved MLG assay of phosphate and a phosphatase to release phosphate from AMP, a spectrometric method successfully estimated the half-inhibition concentrations of papaverine on the recombinant human cyclic nucleotide phosphodiesterase (PDE) isozyme 4 and the mixture of PDE isozymes from rabbit brain. Therefore, the improved MLG assay of phosphate was a favorable and universal technique for developing spectrometric methods for characterizing and screening inhibitors of enzymes that release phosphate during their actions.     

Inhibition of human plasma cholinesterase by malachite green and related triarylmethane dyes: mechanistic implications

The inhibitory effects of the cationic triarylmethane (TAM+) dyes, pararosaniline (PR+), malachite green (MG+), and methyl green (MeG+) on human plasma cholinesterase (BChE) were studied at 25 degrees C in 100 mM Mops, pH 8.0, with butyrylthiocholine as substrate. PR+ and MG+ caused linear mixed inhibition of enzyme activity. The respective inhibitory parameters were K(i) = 1.9 +/- 0.23 microM, alpha = 13 +/- 48, beta = 0 and K(i) = 0.28 +/- 0.037 microM, alpha = 23 +/- 7.4, beta = 0. MeG+ acted as a competitive inhibitor with K(i) = 0.12 +/- 0.017 microM (alpha, infinity, beta, not applicable). The K(i) values were within the same range reported for a number of ChE inhibitors including propidium ion, donepezil, and the phenothiazines, suggesting that TAM+s are active site ligands. On the other hand, the alpha values failed to correlate with values previously reported for a number of ChE inhibitors. It appears that mixed inhibition is the combined result of more than one type of binding and S-I interference. The impact of ligands at the choline-specific and peripheral anionic sites (or, possibly, accessory structural domains) on BChE activity needs to be studied in further detail.     

Serotonin metabolism in rat skin: characterization by liquid chromatography-mass spectrometry

We have recently uncovered the full expression of novel cutaneous serotoninergic and melatoninergic systems in the human and hamster skin. In this work, we have characterized serotonin metabolism in the rat skin using liquid chromatography-mass spectrometry and found that serotonin undergoes acetylation in the presence of acetyl coenzyme A. Inhibition of serotonin acetylation with Cole bisubstrate inhibitor shows that rat skin expresses both arylalkylamine and arylamine N-acetyltransferase activities. The serotonin degradation product-5-hydroxyindole acetic acid is also detected and pargyline (monoaminooxidase inhibitor) suppresses almost completely 5-hydroxyindole acetic acid accumulation. Together with previous data, the present study clearly demonstrates that biotransformation of serotonin in mammalian skin follows two alternate pathways. In the first pathway, serotonin is acetylated by arylalkylamine and arylamine N-acetyltransferases to generate the precursor of melatonin. Alternately, serotonin may undergo oxidative deamination by monoaminooxidase followed by enzymatic degradation by aldehyde dehydrogenase into 5-hydroxyindole acetic acid, which is presumably devoid of biological activity. Thus, the current methodological development of a liquid chromatography-mass spectrometry-based assay allows rapid resolution of the cutaneous metabolism of serotonin.     

Confirmation of malachite green, gentian violet and their leuco analogs in catfish and trout tissue by high-performance liquid chromatography utilizing electrochemistry with ultraviolet-visible diode array detection and fluorescence detection

A sensitive analytical procedure for the confirmation of residues of malachite green (MG), gentian violet (GV) and their leuco analogs (LMG and LGV) in catfish and trout tissue at 10 ng/g is described. Frozen (−20°C) fish fillets were cut into small pieces and homogenized in Waring blendors. The compounds of interest were extracted from 20-g amounts of homogenized fish tissue with acetonitrile-buffer, partitioned against methylene chloride, and isolated with tandem neutral alumina and propylsulfonic acid cation-exchange solid-phase extraction cartridges. Samples of 100 μl (0.8 g equiv.) were chromatographed isocratically in 10 min using an acetonitrile-buffer mobile phase on a short-chain deactivated (SCD) reversed-phase column (150×4.6 mm I.D.) in-line with a post-column oxidation coulometric electrochemical cell (EC), a UV-Vis diode array detector and a fluorescence detector.     

Measurement of 8-hydroxy-2'-deoxyguanosine in DNA by high-performance liquid chromatography-mass spectrometry: comparison with measurement by gas chromatography-mass spectrometry

Measurement of 8-hydroxy-2'-deoxyguanosine (8-OH-dGuo) in DNA by high-performance liquid chromatography/mass spectrometry (LC/MS) was studied. A methodology was developed for separation by LC of 8-OH-dGuo from intact and modified nucleosides in DNA hydrolyzed by a combination of four enzymes: DNase I, phosphodiesterases I and II and alkaline phosphatase. The atmospheric pressure ionization-electrospray process was used for mass spectral measurements. A stable isotope-labeled analog of 8-OH-dGuo was used as an internal standard for quantification by isotope-dilution MS (IDMS). Results showed that LC/IDMS with selected ion-monitoring (SIM) is well suited for identification and quantification of 8-OH-dGuo in DNA at background levels and in damaged DNA. The sensitivity level of LC/IDMS-SIM was found to be comparable to that reported previously using LC-tandem MS (LC/MS/MS). It was found that approximately five lesions per 10(6) DNA bases can be detected using amounts of DNA as low as 2 microgram. The results also suggest that this lesion may be quantified in DNA at levels of one lesion per 10(6) DNA bases, or even lower, when more DNA is used. Up to 50 microgram of DNA per injection were used without adversely affecting the measurements. Gas chromatography/isotope-dilution MS with selected-ion monitoring (GC/IDMS-SIM) was also used to measure this compound in DNA following its removal from DNA by acidic hydrolysis or by hydrolysis with Escherichia coli Fpg protein. The background levels obtained by LC/IDMS-SIM and GC/IDMS-SIM were almost identical. Calf thymus DNA and DNA isolated from cultured HeLa cells were used for this purpose. This indicates that these two techniques can provide similar results in terms of the measurement of 8-OH-dGuo in DNA. In addition, DNA in buffered aqueous solution was damaged by ionizing radiation at different radiation doses and analyzed by LC/IDMS-SIM and GC/IDMS-SIM. Again, similar results were obtained by the two techniques. The sensitivity of GC/MS-SIM for 7,8-dihydro-8-oxoguanine was also examined and found to be much greater than that of LC/MS-SIM and the reported sensitivity of LC/MS/MS for 8-OH-dGuo. Taken together, the results unequivocally show that LC/IDMS-SIM is well suited for sensitive and accurate measurement of 8-OH-dGuo in DNA and that both LC/IDMS-SIM and GC/IDMS-SIM can provide similar results.     

Application of high-performance liquid chromatography-mass spectrometry to detection of diuretics in human urine

A rapid, sensitive and reliable high-performance liquid chromatographic-mass spectrometric method for the detection of 25 diuretics in human urine has been developed. Atmosphere pressure chemical ionization (APCI) and electrospray ionization (ESI) modes were evaluated. A 2-ml volume of urine was extracted under basic conditions and separated on an Agilent Zorbax SB-C(18) column (150 x 2.1 mm, 5 microm). The mobile phase consisted of formic ammonium-formic acid buffer (pH 3.5) and acetonitrile. The effects of capillary temperature, sheath gas pressure and compositions of mobile phase on the sensitivity were studied. The recoveries of most of the diuretics were 75-95%. In the full scan mode, the limits of detection of the 25 diuretics were 0.25-25 ng/ml for APCI and 0.6-250 ng/ml for ESI. Under the optimal conditions, 14 diuretics from authentic urine samples were detected successfully by LC-APCI-MS. To obtain more fragmentation information on the chemical structure for positive confirmation, tandem mass analysis was also investigated.     

Method for liver tissue metabolic profiling study and its application in type 2 diabetic rats based on ultra performance liquid chromatography-mass spectrometry

A protocol for the metabolic profiling of rat liver was developed based on ultra-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS) to explore metabolic state directly. Methanol/water (4:1, v:v) was selected as the optimal extraction solvent. The established method was validated with a linearity over the 10-5000 ng/mL for internal standards (IS) and got an average correlation coefficient of 0.9986. The intra-day and inter-day RSD for most endogenous compounds were below 15%. And the absolute recovery of IS was from 84.8% to 109.1%. Liver tissues from diabetic and control rats were enrolled in the subsequent study to show the usefulness of the method. A clear classification between the control and model animals was achieved, some significant metabolites were successfully filtered. These metabolites reflected the abnormal metabolism of diabetic rats. This initial application indicated that the method is suitable and reliable for liver tissue metabolic profiling. It is expected this protocol could also be extended to metabonomic studies of other tissues.     

Immunoaffinity purification of peptide hormones prior to liquid chromatography-mass spectrometry in doping controls

For most peptide hormones prohibited in elite sports the concentrations in plasma or urine are very low (pg/mL). Accordingly, hyphenated purification and enrichment steps prior to mass spectrometric detection are required to obtain sufficient doping control assays. Immunoaffinity purification in combination with nano-scale liquid chromatography coupled to high resolution/high accuracy mass spectrometry was found to have the potential of providing the necessary sensitivity and unambiguous specificity to produce reliable results. With the presented methodology 12 prohibited peptides (porcine insulin, Novolog, Apidra, Lantus DesB30-32 metabolite, Humalog and human insulin, Synacthen (synthetic ACTH analogue), luteinizing hormone-releasing hormone (LH-RH), growth hormone releasing hormone (GH-RH(1-29)) and CJC-1295 (GH-RH analogue), LongR(3)-IGF-1 and IFG-1) were simultaneously purified from plasma/serum or urine. With limits of detection for each target compound ranging in the low pg/mL level (urine), the method enables the determination of urinary peptides at physiologically relevant concentrations. For each class of peptides an appropriate antibody and a respective internal standard was implemented ensuring robust analysis conditions. Due to the fast and simple sample preparation procedure (～25 samples per day) and the fact that all materials are commercial available, the implementation of the methodology to laboratories from other analytical fields (forensics, pharmacokinetic sciences, etc.) is enabled.     

Determination of myo-inositol in biological samples by liquid chromatography-mass spectrometry

A high-performance liquid chromatographic method using liquid-liquid extraction was developed for the determination of 1-(3-fluoro-4-hydroxy-5-mercaptomethyl-tetrahydrofuran-2-yl)-5-methyl-1H-pyrimidine-2,4-dione (l-FMAUS; I) in rat plasma and urine. A 100 microl aliquot of distilled water containing l-cysteine (100 mg/ml) was added to a 100 microl aliquot of biological sample. l-Cysteine was employed to protect binding between the 5'-thiol of I and protein in the biological sample. After vortex-mixing for 30s and adding a 50 microl aliquot of the mobile phase containing the internal standard (10 microg/ml of 3-aminophenyl sulfone), 1 ml of ethyl acetate was used for extraction. After vortex-mixing, centrifugation, and evaporating the ethyl acetate, the residue was reconstituted with a 100 microl aliquot of the mobile phase. A 50 microl aliquot was injected onto a C(18) reversed-phase column. The mobile phases, 50 mM KH(2)PO(4) (pH = 2.5):acetonitrile (85:15, v/v) for rat plasma and 50 mM KH(2)PO(4) (pH 2.5):acetonitrile:methanol (85:10:5, v/v/v) for urine samples, were run at a flow-rate of 1.2 ml/min. The column effluent was monitored by an ultraviolet detector set at 265 nm. The retention times for I and the internal standard were approximately 9.7 and 12.5 min, respectively, in plasma samples and the corresponding values in urine samples were 16.8 and 14.9 min. The quantitation limits of I in rat plasma and urine were 0.1 and 0.5 microg/ml, respectively.     

Determination of Simvastatin in human plasma by liquid chromatography-mass spectrometry

A simple, sensitive and selective liquid chromatography coupled with electrospray ionization mass spectrometry (LC/ESI/MS) method for the determination of simvastatin (I) has been developed. After extraction by ethyl acetate, using lovastatin (II) as internal standard, solutes are separated on a C(18) column with a mobile phase consisting of methanol-water (9:1). Detection is performed on an atmospheric pressure ionization single quadruple mass spectrometer equipped with an ESI interface and operates in positive ionization mode. Simvastatin quantification was realized by computing peak area ratio (I/II) of the extracts analyzed in SIM mode (m/z: 441 and m/z: 427 for I and II, respectively) and comparing them with calibration curve (r=0.9997). Accuracy and precision for the assay were determined by calculating the intra-batch and inter-batch variation at three concentrations 0.1, 5.0, 10.0 ng/ml; the intra batch relative standard deviation (RSD) was less than 10% and ranged from 1.8 to 8.5%, respectively; the inter-batch RSD was less than 20% and ranged from 4.1 to 16.5%. The limit of detection was 0.05 ng/ml.     

Comparative urine analysis by liquid chromatography-mass spectrometry and multivariate statistics: method development, evaluation, and application to proteinuria

We describe a platform for the comparative profiling of urine using reversed-phase liquid chromatography-mass spectrometry (LC-MS) and multivariate statistical data analysis. Urinary compounds were separated by gradient elution and subsequently detected by electrospray Ion-Trap MS. The lower limit of detection (5.7-21 nmol/L), within-day (2.9-19%) and between-day (4.8-19%) analytical variation of peak areas, linearity (R2: 0.918-0.999), and standard deviation for retention time (<0.52 min) of the method were assessed by means of addition of seven 3-8 amino acid peptides (0-500 nmol/L). Relating the amount of injected urine to the area under the curve (AUC) of the chromatographic trace at 214 nm better reduced the coefficient of variation (CV) of the AUC of the total ion chromatogram (CV = 10.1%) than relating it to creatinine (CV = 38.4%). LC-MS data were processed, and the common peak matrix was analyzed by principal component analysis (PCA) after supervised classification by the nearest shrunken centroid algorithm. The feasibility of the method to discriminate urine samples of differing compositions was evaluated by (i) addition of seven peptides at nanomolar concentrations to blank urine samples of different origin and (ii) a study of urine from kidney patients with and without proteinuria. (i) The added peptides were ranked as highly discriminatory peaks despite significant biological variation. (ii) Ninety-two peaks were selected best discriminating proteinuric from nonproteinuric samples, of which 6 were more intense in the majority of the proteinuric samples. Two of these 6 peaks were identified as albumin-derived peptides, which is in accordance with the early rise of albumin during glomerular proteinuria. Interestingly, other albumin-derived peptides were nondiscriminatory indicating preferential proteolysis at some cleavage sites.