Liquid Chromatography-ionspray Mass Spectrometry Research Articles

Simultaneous determination of estramustine phosphate and its four metabolites in human plasma by liquid chromatography-ionspray mass spectrometry.

A sensitive and selective method, using liquid chromatography-ionspray mass spectrometry, was developed and validated for the simultaneous determination of Estracyt (estramustine phosphate) and its four metabolites, estramustine, estromustine, estrone and estradiol, in human plasma. Deuterated internal standards were available for all analytes. The five compounds were extracted from plasma by protein precipitation with acetonitrile. The chromatographic separation was performed using a Zorbax SB C18, (150 x 4.6 mm i.d., 5 microm) reversed-phase column under gradient conditions with a mobile phase containing 2 mm ammonium acetate buffer (pH 6.8) and acetonitrile. MS detection was by electrospray ionization with multiple reaction monitoring in the positive ion mode for estramustine phosphate, estromustine and estramustine, and in the negative ion mode for estrone and estradiol. The limit of quantitation was 10 ng/mL for estramustine phosphate, 3 ng/mL for estromustine, estramustine and estrone and 30 ng/mL for estradiol. Linearity was verified from these LLOQs up to about 4000 ng/mL for the parent drug and 2000 ng/mL for the metabolites. Inter-day precision and accuracy values were all less than 15%. This assay was applied successfully to the routine analysis of human plasma samples collected in cancer patients administered estramustine phosphate intravenously.

Simultaneous determination of JTT-501 and its main metabolite in human plasma by liquid chromatography–ionspray mass spectrometry

An LC–MS–MS analytical method was developed for the determination of a new antidiabetic agent, JTT-501 and its main metabolite (JTP-20604) in human plasma. The compounds were isolated from plasma by protein precipitation before analysis by HPLC with atmospheric pressure positive ionisation MS–MS detection. An isotopically labelled analog of JTT-501 was used as the internal standard. Linearity was demonstrated over the calibration range of about 5–10 000 ng/ml for both compounds. The assay was validated with respect to accuracy, precision and analyte stability. This method was used for the determination of plasma concentrations for the two compounds in a clinical tolerability study. A cross-validation exercise between two different mass spectrometers, used for the determination of clinical samples, is also reported.

Quantification of busulfan in plasma by liquid chromatography–ion spray mass spectrometry: Application to pharmacokinetic studies in children

Optimisation of busulfan dosage in patients undergoing bone marrow transplantation is recommended in order to reduce toxic effects associated with high drug exposure. A new method was developed coupling liquid chromatography with mass spectrometry (LC–MS) and was validated for the determination of busulfan concentrations in plasma. Recovery was 86.7%, the limit of detection was 2.5 ng/ml and linearity ranged from 5 to 2500 ng/ml. The correlation between the busulfan concentrations measured by our previously published HPLC–UV method and the new HPLC–MS method was highly significant ( P<0.0001). Sample volume was reduced and the method was rapid, sensitive and less expensive than the methods previously used in our laboratory. This method was used to determine the pharmacokinetic parameters of busulfan after the first administration of 1 mg/kg orally, in 13 children receiving the drug as part of the preparative regimen for bone marrow transplantation. Our results were similar to previously reported data. They showed that the apparent oral clearance of busulfan was 0.299±0.08 l/h/kg, and that it was significantly higher ( P=0.02) in patients below the age of 5 years than in older children.

Sensitive and specific multiresidue methods for the determination of pesticides of various classes in clinical and forensic toxicology

Original and sensitive multiresidue methods are presented for the detection and quantitation, in human biological matrices, of 61 pesticides of toxicological significance in human. These methods involved rapid solid-phase extraction using new polymeric support (HLB and MCX) OASIS ® cartridges. Gas chromatography-mass spectrometry (GC-MS) was used for volatile (organophosphate, organochlorine, phtalimide, uracil) pesticides and liquid chromatography-ionspray ®-mass spectrometry (LC-MS) for thermolabile and polar pesticides (carbamates, benzimidazoles). Acquisition was performed in the selected ion monitoring (SIM) mode. Extraction recovery varied owing to the nature of pesticides, but was satisfactory for all. Limits of detection (LODs) and limits of quantitation (LOQs) ranged, respectively, from 2.5 to 20 and from 5 to 50 ng/ml. An excellent linearity was observed from LOQs up to 1000 ng/ml for all the pesticides studied. The proposed procedures yielded reproducible results with good inter-assay accuracy and precision. A few cases of intoxication are presented to demonstrate the diagnostic interest of these methods: in two cases were determined lethal concentrations of endosulfan and carbofuran; in four other cases, the procedures helped diagnose intoxication with, respectively, parathion-ethyl, the association of bromacil and strychnine, bifenthrin and aldicarb.

Identification of selected psychopharmaceuticals and their metabolites in hair by LC/ESI-CID/MS and LC/MS/MS

Hair samples of patients of psychiatry and hair samples of suicide cases were analysed by liquid-chromatography/ionspray-mass spectrometry (LC/MS) for antidepressants and neuroleptics. Electrospray ionisation (ESI) with in-source collision induced dissociation (ESI/CID) and tandem-mass spectrometry (MS/MS) were used for drug and metabolite identification. Mass spectra library searching was performed using an ESI/CID mass spectra library and a MS/MS spectra library. Furthermore, extracted ion chromatograms were used for the detection of N-desmethyl-metabolites, which were also identified by their fragment-ion spectra. Three examples using these methods are shown: The tricyclic antidepressant maprotiline, the selective serotonin receptor inhibitor (SSRI) citalopram and their desmethylmetabolites as well as the neuroleptic pipamperone were detected and identified in hair extracts. For extraction powdered hair was treated by ultrasonication in methanol and solid-phase extraction was used for sample clean-up prior to LC/MS or MS/MS analysis. These examples demonstrate the power of LC/MS and LC/MS/MS for the detection and identification of drugs in hair extracts using full-scan mode and ESI/CID with library searching or using highly selective LC/MS/MS-analysis with library searching or in multiple reaction monitoring mode.

Identification of ten corticosteroids in human hair by liquid chromatography–ionspray mass spectrometry

This paper describes a screening procedure based upon high-performance liquid chromatography–ionspray mass spectrometry for the identification of ten corticosteroids in human hair: triamcinolone, prednisolone, prednisone, methylprednisolone, cortisone, cortisol, beta- and dexa-methasone, flumethasone and beclomethasone. Hair strands were washed in methylene chloride, pulverized in a ball mill and 50 mg of the powdered hair were incubated in 1 ml Soerensen buffer, pH 7.6 for 16 h at 40°C, in presence of 50 ng cortisol-d3 used as internal standard. Purification of the incubation medium was achieved on SPE C18 Isolute extraction columns. The eluates were evaporated to dryness and resuspended in 30 μl MeOH before analysis by HPLC–IS-MS in positive and negative modes of detection. The validation parameters were found satisfactory for a corticosteroid screening procedure. The correlation coefficient of the calibration curve ranged from 0.939 to 0.997, showing linearity between 0.1 and 10 ng/mg, excepted for beclomethasone which was between 0.2 and 10 ng/mg. Extraction recovery at 4 ng/mg ranged from 43.2 to 85.7%. Repeatability (CV values) at 4 ng/mg ranged from 6.1 to 17.5%. The limits of detection ranged from 0.03 to 0.17 ng/mg for a signal-to-noise ratio of 2. The detection of prednisone and beclomethasone in three hair specimens obtained from forensic and clinical cases have documented corticosteroids incorporation into human hair.

Validation of a liquid chromatography ionspray mass spectrometry method for the analysis of flavanones, flavones and flavonols.

The application of liquid chromatography/mass spectrometry (LC/MS) with a TurboIonspray (TIS) interface was investigated as a new method for the analysis of flavonoids. Eleven compounds belonging to three different classes of flavonoids were studied: eriocitrin, neoeriocitrin, naringin, narirutin, hesperidin, neohesperidin (flavanone glycosides), quercetin, kaempferol, galangin (flavonol aglycones), chrysin, apigenin (flavone aglycones). Chromatographic separations were performed under reversed-phase conditions using a C18 narrow-bore LC column; a mixture of an aqueous solution of formic acid (pH 2.4) and acetonitrile was used as the mobile phase. Isocratic elution was operated in the case of flavanones, whereas gradient elution was used for the simultaneous separation of flavones and flavonols. The adaptability of TIS to high flow applications allows the use of LC eluent flow rates at 200 µL/min without post-column splitting. Qualitative analysis was performed in negative-ion (NI) full-scan mode, whereas response linearity, detection limits and precision of the method were studied under NI selected ion monitoring (SIM) conditions. Characterization of isomers differing in the glycosylation was found to be possible on the basis of different mass spectra. Detection limits in the low-ng range (0.08-0.4 ng) were found, about twenty-fold lower than those reported previously. The method was applied to identify and determine the content of flavonoids in an orange juice sample. Copyright 1999 John Wiley & Sons, Ltd.

Determination of parts per trillion level of carboxylic degradation products of linear alkylbenzenesulfonates in coastal water by solid-phase extraction followed by liquid chromatography/ionspray/mass spectrometry using negative ion detection

Long-chain sulfophenyl carboxylate compounds (SPC) with more than five carbon atoms, which are degradation products of linear alkylbenzenesulfonates (LAS), have been isolated by solid-phase extraction (SPE) followed by identification with liquid chromatography/ ionspray—mass spectrometry (LC/ISP-MS). The isolation procedure involved extraction in a C18 minicolumn and subsequently in a strong anionic exchange (SAX) column, the final determination was accomplished by Negative Ion LC/ISP/MS using a two step approach. First, a chromatographic run at a cone voltage of 80 V was performed. At this voltage the major fragment ion corresponded either to m/z 183 (C11 SPC and LAS) or to m/z 170 (C6SPC to C10SPC), and permitted the confirmation of LAS and SPC. The second step involved an extraction cone voltage of 20 V. In this case [M-H]− ion was the base peak for all the studied compounds (LAS and SPC) and was used as a quantitative ion. The linear range of the proposed method varied from 0.4 to 10 ppb with a limit of detection ranging from 2–20 ppt when 250 mL of coastal water were preconcentrated. The present method represents an important advancement for the unequivocal trace determination of the polar degradation products of LAS, SPC, since current established protocols involve either the use of derivatization/gas chromatography-mass spectrometry and/or liquid chromatography with UV detection. By using the protocol described the direct confirmation and quantitative trace determination of both LAS and their polar metabolites SPC is feasible.

Determination of nitrofuran residues in avian eggs by liquid chromatography–UV photodiode array detection and confirmation by liquid chromatography–ionspray mass spectrometry

A high-performance liquid chromatographic–UV–Vis photodiode-array detection (HPLC–DAD) method for the determination of nitrofuran residues, nitrofurazone, furazolidone and furaltadone, in chicken eggs is described. Confirmation of the identity of nitrofurazone, furazolidone and furaltadone was performed by high-performance liquid chromatography–mass spectrometry (HPLC–MS) using an atmospheric pressure ionization (API) source and an ionspray interface. The nitrofuran residues were extracted from eggs with acetonitrile and the extracts purified by liquid–liquid partitioning. Analytes were chromatographed isocratically with an octadecylsilyl (ODS) column and an UV–Vis detector set at 362 nm and identified by comparing the retention times and UV–Vis spectra of the sample peaks with the reference compounds. The HPLC–DAD limit of detection based on a signal-to-noise ratio (S/N) of 3, was estimated to be 2.5 μgkg−1 for nitrofurazone and furazolidone and 5.0 μgkg−1 for furaltadone. The ionspray HPLC–MS was carried out on the purified extracts. The HPLC–MS method involved the separation of analytes on a C18 column with acetonitrile–water (50:50, v/v), containing 1 mM ammonium acetate and 0.025% acetic acid, with selected-ion monitoring (SIM) of only protonated molecules, [M+H]+ of the analytes. The overall average recovery in nitrofuran fortified eggs was 85.3±3.8% for nitrofurazone, 88.1±3.9% for furazolidone and 87.1±3.7% for furaltadone. The HPLC–MS limit of detection, based on a S/N of 3, was estimated to be 3.2, 1.6 and 1.0 μgkg−1 for nitrofurazone, furazolidone and furaltadone, respectively. HPLC–MS has shown itself to be a sensitive, selective and rapid method and was successfully used for the confirmation analysis of nitrofurazone, furazolidone and furaltadone in avian eggs for regulatory purposes.

Determination of traces of pesticides in water by solid-phase extraction and liquid chromatography–ionspray mass spectrometry

A multi-residue analytical method for six pesticides (atrazine, hydroxyatrazine, carbofuran, promecarb, linuron and monolinuron) in drinking water has been developed. The method combines liquid chromatography and mass spectrometry using an ionspray interface. The linearity domain, as well as the limits of detection and quantification, were determined for each compound. Although satisfactory performance could be achieved, present drinking water regulations (0.1 μg l−1 for single pesticide) requires a pre-concentration step. This was performed using solid-phase extraction with octadecyl-bonded silica cartridges. The analytical procedure was tested on water samples spiked at the 0.04 and 0.08 μg l−1 levels, and allowed the determination of the investigated pesticides (except hydroxyatrazine) at these trace concentrations.

High-performance liquid chromatography–ionspray mass spectrometry for the specific determination of digoxin and some related cardiac glycosides in human plasma

An original method based upon high-performance liquid chromatography coupled to ionspray mass spectrometry (HPLC–ISP-MS) has been developed for the identification and quantification in plasma of several cardiac glycosides, namely digoxin, digitoxin, lanatoside C and acetyldigitoxin. After single-step liquid–liquid extraction by chloroform–2-propanol (95:5, v/v) at pH 9.5 using oleandrin as an internal standard, solutes are separated on a 4 μm NovaPak C 18 (Waters) column (150×2.0 mm, I.D.), using a gradient of acetonitrile–2 m M NH 4COOH, pH 3 buffer (flow-rate 200 μl/min, post-column split 1:3). Detection is done by a Perkin-Elmer Sciex API-100 mass analyzer equipped with an ISP interface. In most instances the major ion observed is not [M+H] + as expected, but [M+NH 4] +. The mean retention times (min) are: lanatoside C, 5.74; digoxin, 6.00; digitoxin, 8.08, oleandrin, 8.30, acetyldigitoxin, 8.66 and 9.01 (isomers α and β, respectively). The lower limits of detection in single ion monitoring mode range from 0.15 ng/ml (α- and β-acetyldigitoxin) to 0.60 ng/ml (lanatoside C), making the method less sensitive than radioimmunoassay, whereas it is much more specific.

Study of the acetaldehyde induced polymerisation of flavan-3-ols by liquid chromatography-ion spray mass spectrometry

In some fruits, the loss of astringency has been attributed to the insolubilization of recombinated tannins, formed by reaction with acetaldehyde. This route has been investigated by on-line coupling of liquid chromatography with ion spray mass spectrometry (LC-ISP-MS). The results reported herein indicate that LC-ISP-MS can be successfully applied to tannin polymers and is very helpful in their identification. It also allows direct monitoring of the progress of polymerisation reactions. The mechanism of the acetaldehyde-induced polymerisation of flavan-3-ols has thus been demonstrated.

Automated sample preparation with extraction columns followed by liquid chromatography-ionspray mass spectrometry interferences, determination and degradation of polar organophosphorus pesticides in water samples

The determination of polar and/or thermally labile organophosphorus pesticides trichlorfon, dichlorvos, dimethoate, oxydemeton-methyl, mevinphos ( cis and trans), demeton-S-methyl, fenamiphos, fenitrothion, fenthion and diazinon in water samples was investigated using solid-phase extraction followed by liquid chromatography-ionspray mass spectrometry (LC-ISP-MS). Pesticides were spiked at 0.2 μg/l in ground water samples and 200 ml were preconcentrated by using an ASPEC XL system. To improve the recovery of the most volatile pesticides such as dichlorvos, a further drying step with a Baker Spe 12 G apparatus with the vacuum set at 15 p.s.i. was included. A further evaporation step was applied with a gentle stream of nitrogen. A variety of materials (Amberchrom, LiChrolut EN, cyclohexyl, SDB, C 18 and Isolute ENV) were used for the isolation of the target pesticides. The elution step took place at 1 ml/min, with a 5-min contact time between the eluent and the sorbent. LC-ISP-MS permitted the identification of diethyl and di- n-butyl phthalates present as impurities in the cartridge materials. The presence of transformation products of the pesticides, e.g., fenthion sulfoxide, was also observed. The influence of pH and the water matrix (distilled and ground water) on the extraction efficiency was examined. The use of LC-ISP-MS permitted cis- and trans-mevinphos to be distinguished in the water sample owing to the presence of a specific ion at m z 99 . the limits of detection of the overall method were in the range 0.01–0.20 μg/1 using LC-ISP-MS with selected-ion monitoring.

Determination of BMS-186318 in dog, rat and monkey plasma by liquid chromatography-ionspray mass spectrometry

BMS-186318 is a member of the recently discovered “aminodiol” class of HIV protease inhibitors. A simple but sensitive method was developed for the determination of BMS-186318 in dog plasma and then applied to monkey and rat plasma. The compound was extracted from dog plasma with methyl tert-butyl ether at basic pH. The dried extract was reconstituted in mobile phase and injected into a 150 × 2.1 mm i.d. Zorbax Rx-C 18 HPLC column. A portion of the effluent was directed into the LC-ionspray MS system, where the [M + H] + ion of the secondary amine compound was monitored. The HPLC conditions were chosen in order to achieve a short run time and large sample throughput, with both analyte and internal standard eluting within 1.5 min. The liquid-liquid extraction procedure provided very clean extracts so that sufficient signal-to-noise ratio was obtained with single-stage mass spectrometry instead of the more costly tandem mass spectrometry. The required lower limit of quantitation of 2.5 ng ml −1 was easily achieved. The method has also been validated for BMS-186318 in monkey plasma without modification. The method has been modified for rat plasma. Owing to irreproducibility observed when applying the liquid-liquid extraction method to rat plasma, a solid-phase extraction method was developed. The addition of phenylmethylsulfonyl fluoride was necessary to stabilize BMS-186318 in rat blood and plasma.

The extracellular PI-type proteinase of Lactococcus lactis hydrolyzes beta-casein into more than one hundred different oligopeptides.

The peptides released from beta-casein by the action of PI-type proteinase (PrtP) from Lactococcus lactis subsp. cremoris Wg2 have been identified by on-line coupling of liquid chromatography to mass spectrometry. After 24 h of incubation of beta-casein with purified PrtP, a stable mixture of peptides was obtained. The trifluoroacetic acid-soluble peptides of this beta-casein hydrolysate were fractionated by high-performance liquid chromatography and introduced into the liquid chromatography-ion spray mass spectrometry interface. Multiply charged ions were generated from trifluoroacetic acid-soluble peptides under low nozzle voltage conditions, yielding the MH+ mass of each eluted peptide. All peptides corresponding to each of the MH+ calculated masses were determined. In those cases in which different peptides were possible, further identification was achieved by collision-induced dissociation under higher nozzle voltage conditions. Hydrolysis of beta-casein by PrtP was observed to proceed much further than reported previously. More than 40% of the peptide bonds are cleaved by PrtP, resulting in the formation of more than 100 different oligopeptides. With the exception of Phe, significant release of amino acids or di- and tripeptides could not be observed. Interestingly, one-fifth of the identified oligopeptides are small enough to be taken up by the oligopeptide transport system. Uptake of these peptides could supply L. lactis with all amino acids, including the essential ones, indicating that growth of L. lactis might be possible on peptides released from beta-casein by proteinase only.

Evaluation of liquid chromatography—ion spray mass spectrometry for the determination of substituted benzoic acids and their glycine conjugates

Liquid chromatography combined with pneumatically assisted electrospray (ion spray) mass spectrometry was evaluated for the characterization of low-molecular-mass compounds such as a variety of substituted benzoic acids and their glycine conjugates. Each substituted benzoic acid and its glycine conjugate formed with rat liver mitochondria were separated with a conventional C 8 packed column or a semimicro C 8 column using three mobile phases. The positive- and negative-ion mass spectra of the glycine conjugates gave abundant [M + H] + and [M − H] − ions, respectively. However, the positive-ion mass spectra of the acids were dominated by [M + H − H 2O] + ions, with the exception of [M + H] + ions for acids having an amino, an acetylamino or a dimethylamino group. All of the negative-ion mass spectra of acids gave dominant [M − H] − ions. The negative-ion mass spectra of acids with halogen, nitro and cyano groups showed the ions equivalent to the mass of each functional group. Further, the alkoxyl group gave a characteristic fragmentation representing loss of the alkyl moiety. The abundances of the fragment ions due to the functional groups gave information on the position of the ring substituents in the negative-ion mass spectra. The positive- and negative-ion mass spectra of the glycine conjugates revealed the presence of glycine.

Characterization of Tryptic Peptides of a Potent Growth Hormone Releasing Hormone Analog by Reversed Phase High Performance Liquid Chromatography-Ionspray Mass Spectrometry

Abstract A highly reproducible tryptic digestion procedure was developed for the characterization of a potent growth hormone releasing hormone (GHRH) analog, para-methylhippuroyl derivative of the N-terminus of a precursor porcine-GHRH (2–76) (pMHpGHRH) derived by recombinant DNA technology. Tryptic digestion can be completed within one hour at room temperature. All tryptic peptides can be separated by reversed phase high performance liquid chromatography (RPHPLC) on a Vydac C18 protein column with trifluoroacetic acid(TFA)-acetonitrile (ACN) gradient elution. Eleven single fragments obtained in tryptic peptide mapping have been identified by coupled Ionspray mass spectrometry. Combined fragments T2-3 and T4-5 were also observed and verified by Ionspray mass spectrometry and microsequencing analysis. A stability study indicated that the digestion solution will remain stable for 9 days at 5 °C. The retention time variations for the tryptic peptides found to be 1.9% relative standard deviation (RSD).

Structural characterization of the decomposition products of salbutamol by liquid chromatography—ionspray mass spectrometry

Liquid chromatography—ionspray mass spectrometry was used to elucidate the structures of the decomposition products of salbutamol. The best sensitivity in mass spectrometry was achieved by using a mixture of acetonitrile and ammonium formate (10 mM, pH 3.3) as the mobile phase in liquid chromatography. Fragmentation of the compounds was obtained by increasing the nozzle voltage in the first vacuum stage of the mass spectrometer. Tentative structure elucidation showed that both acidic and basic decomposition products are formed from salbutamol.

Identification with liquid chromatography-ionspray mass spectrometry of the metabolites of the enantiomers N-methyl dextrorphan and N-methyl levorphanol after rat liver perfusion.

To gather more information on stereochemical factors in the hepatic disposition of organic cations, mass spectrometry coupled to liquid chromatography was used to determine the identity of the metabolites excreted in bile after isolated rat liver perfusions with the quaternary ammonium derivatives of the enantiomeric drugs dextrorphan and levorphanol. Ionspray mass spectrometry was chosen for its soft ionization and absence of thermal degradation of labile compounds. The drugs were labelled with a stable (2H) isotope and mixed with unlabelled drugs to create an artificial isotope pattern in the mass spectrum and facilitate the recognition of unknown metabolites. In mass spectra that were recorded under normal conditions, fragmentation was absent and metabolites of N-methyl dextrorphan and N-methyl levorphanol were visible as parent-ion 'doublets'. Collision-induced fragmentation studies were performed to support the identification of the metabolites. For N-methyl dextrorphan the glucuronide, the glutathione conjugate and the glucuronide of the N-demethylated metabolite were found in bile. For N-methyl levorphanol the glucuronide, the glutathione conjugate, the sulphate conjugate and the glucuronide of a hydroxylated N-methyl levorphanol were excreted in bile. Thus a remarkable stereoselectivity occurs in the metabolism of these quaternary ammonium compounds in the rat liver.

Microbore high performance liquid chromatography–ion spray mass spectrometry for the determination of peptides

AbstractMicrobore high performance liquid chromatography‐ion spray mass spectrometry is demonstrated for the determination of peptides. The ion spray mass spectra of peptides contained primarily only (M + nH)n+ ions (n is the degree of protonation). The linearity for a dilution series of leucine enkephalin was determined to be 4.9 orders of magnitude (r = 0.990), from 29 fmol to 90 pmol injected, and the smallest discernible signal (S/N of 3/1) was 65 amol. The tandem mass spectrometry daughter‐ion mass spectrum of the multiply‐charged molecular ion of dynorphin 1–9 exhibited daughter ions consistent with the sequence of the peptide. The micro‐LC gradient elution separation for a tryptic digest of recombinant bovine somatotropin was acquired. The mass spectra obtained from the components in the tryptic digest were consistent with previously identified peptides. The mass spectrum of the tryptic fragment ISLLLIQSWLGPLQFLSR (MW 2083) contained multiply‐charged ions that made it possible to detect this fragment even though its molecular weight is beyond the mass range of the mass spectrometer.