Mass Spectrometric Method For Determination Research Articles

Liquid chromatography with tandem mass spectrometric method for determination of 52 antibiotics in human whole blood and urine and application to forensic cases.

A rapid and reliable method was developed and validated for the simultaneous analysis of 52 antibiotics (cephalosporins, penicillins, carbapenems, lincosamides, quinolones, nitroimidazoles, macrolides, sulfonamides, tetracyclines, glycopeptide) in urine and whole blood by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). Analytes were extracted by dilution or protein precipitation and analyzed on an Agilent 1260 HPLC system coupled to an Agilent 6470 Triple Quadrupole Mass Spectrometer. The method attended method validation criteria. The limits of detection were equal or lower than 2.0ng/mL, whereas the limits of quantification ranged from 0.1 to 10.0ng/mL, from 0.1 to 5.0ng/mL, in urine and whole blood, respectively. For all analytes, the bias and intra- and inter-day precision values were less than 14.7%. The ranges of recovery values of all antibiotics were 76.5-124.5% in whole blood and 76.3-121.8% in urine, values of the effects were lower than 25% in two matrices. No evidence of carryover was observed. The study of sample stability showed that almost all analytes were stable at 24°C for 24h, all analytes were stable at -20°C for 14 days and at -80°C for 30 days. Freeze-thaw cycles stability showed that antibiotics were stable except for imipenem. Autosampler stability study showed that all analytes were stable for 24h, except for imipenem and amoxicillin. Applicability was proven by analyzing authentic whole blood (n = 86) and urine (n = 79) samples from patients under antibiotics treatment. Therefore, this method was applied to the analysis 3 forensic allergy cases, which were positive for at least one analyte. A simple, sensitive and high-throughput method for the simultaneous determination of different classes of antibiotics in urine and whole blood samples was developed and applied. This sensitive method was successfully applied to forensic cases.

A Sensitive Ultra-performance Liquid Chromatography-Tandem Mass Spectrometric Method for Determination of Secalonic Acid F in Rat Plasma and Its Application to Pharmacokinetic Study.

Secalonic acid F (SAF) is a fungal secondary metabolite exhibited interesting pharmacological effect. In this study, a simple and sensitive ultra-performance liquid chromatography-tandem mass spectrometric (UPLC-MS/MS) method was developed and validated for the determination of SAF in rat plasma. Emodin was selected as the internal standard (IS), and plasma samples were prepared by liquid-liquid extraction with ethyl acetate. Chromatographic separation was operated on an Agilent SB-C18 column, and the mobile phase was a mixture of 0.5% formic acid in water and methanol (V:V =20:80) at a flow rate of 0.3mL/min. Detection was carried out with a 6460 triple-quadrupole mass spectrometer using electrospray ionization in the multiple-reaction monitoring mode. The MS/MS ion transitions monitored were m/z 639.3→415.4 and 269.0→225.1 for SAF and IS, respectively. Results showed the calibration curve of SAF was linear in the range of 2-500ng·mL-1 with the correlation coefficient > 0.99. The matrix effect, extraction recovery, dilution effect, intraday and inter-day precision and accuracy were all in acceptable limits. The analytes were also stable under different conditions. The validated method was successfully applied to a pharmacokinetic study after oral administration in Sprague-Dawleyrats at a dose of 10mg/kg.

Liquid chromatography with tandem mass spectrometric method for determination of 425 drugs and poisons in dried blood spots and application to forensic cases.

An analytical method using liquid chromatography with tandem mass spectrometry (LC-MS/MS) was established and validated for screening 425 drugs and poisons in dried blood spots (DBSs). Blood (20 μL) was spotted on Whatman FTA™ classic cardto prepare DBS sample, then extracted with 150 μL methanol and analyzed by LC-MS/MS using a multiple reaction monitoring method. The limit of detection of the compounds were 0.1-10ng/mL. The values for recovery and matrix effect were 40.3-114.9% and 40.2-118.4%, respectively. This method was successfully applied to DBS samples from 105 humans suspected of drug poisoning, which was stored for 3-5years at room temperature. Thirty-three kinds of drugs, including benzodiazepines, antipsychotics, antidepressants, antipyretic analgesics, non-steroidal anti-inflammatory drugs, antibiotics, antiepileptic drugs, new psychoactive drugs were confirmed in 102 cases, while no compound was detected in the other 3 cases. Estazolam, a benzodiazepine widely used in clinical practice as a sedative, hypnotic, and anti-anxiety drug, was the most frequently detected substance, occurring in 34.2% of the cases. Most drugs in DBS could still be detected after storage for 3-5years, but ambroxol, zopiclone, carbofuran, chlorpyrifos, and valproic acid were not detectable after 3-5years of storage at room temperature. The components measured in DBS were consistent with those measured in whole blood at the collection time, thereby confirming that DBS samples have the advantage of stable storage at room temperature.

A sensitive liquid chromatography-tandem mass spectrometric method for determination of five β-blockers after labeling with either hydrazonoyl chloride or dansyl chloride reagent

A sensitive liquid chromatography-tandem mass spectrometry (LC-MS) method was developed for the screening of five β-blockers (BBs), including atenolol, metoprolol, bisoprolol, propranolol, and betaxolol, in rabbit plasma. An inhouse prepared hydrazonoyl chloride compound (UOSA54) and dansyl chloride (DNS) were successfully coupled with BBs via the amino functional group and analyzed by LC-MS/MS. The excess hydrazonoyl chloride was characterized by a negligible ionization suppression at the electrospray-ionization (ESI) source, which enables the analysis of BBs at a low concentration level. The relative ESI-MS response of derivatized to underivatized BBs was enhanced 2.3 to 3.7-fold. The developed method could be applied for screening of BBs in samples by searching the most abundant MS product ions, including m/z 169 and 211, in addition to the precursor ion and the cleavage of ether moiety. The method could be applied for trace analysis and screening of BBs abuse. The use of UOSA54 was adventitious over dansylated derivatives because of minimal reaction by-products and the negligible ionization suppression effect. The extraction efficiency of BBs from rabbit plasma was improved to reach 77.5–93.9% using tert-butylamine and Chromabond® C18ec-100 mg column. The optimal reaction conditions were optimized and validated. The linear range of analyzed BBs in rabbit plasma was within the range of 0.1 to 25.0 ng/mL, while the limit of quantification (LO Q) was ranged from 0.10 to 0.25 ng/mL.

Molybdenum and lanthanum as alternate burn-up monitors – development of chromatographic and mass spectrometric methods for determination of atom percent fission

Abstract A rapid high performance liquid chromatography (HPLC) and thermal ionisation mass spectrometric (TIMS) methods have been developed for the separation and estimation of fission product elements molybdenum and lanthanum for the burn-up measurements on the dissolver solution of Indian pressurised heavy water reactor (PHWR) spent fuel. Reverse phase chromatography method was developed to separate molybdenum from dissolver solution using mandelic acid as mobile phase and a dynamic ion exchange chromatography technique was used for the separation of lanthanum as well as neodymium from a dissolver solution. Sample loading methods which resulted in enhanced ionisation efficiency have been developed for the TIMS analysis of HPLC separated molybdenum and lanthanum fractions. Ascorbic acid mixed with silicic acid in HCl medium was used for loading the molybdenum on to a rhenium filament to obtain stable and intense ion beam. A novel sample loading method for lanthanum in which a mixture of graphite + boric acid (H3BO3) + silica gel was employed to achieve enhanced and steady ion beam formation of LaO+. Concentrations of species of interest were determined employing suitable spikes by isotope dilution mass spectrometry (IDMS) method. The developed methods were adopted for PHWR dissolver solution to establish molybdenum and lanthanum as alternate burn-up monitors. The burn-up data obtained were compared with the well established method of neodymium as fission product monitor. This is a first study of its kind where the data obtained by using molybdenum and lanthanum as fission product monitors were compared with that obtained by Nd-148 method.

Development and Validation of a Rapid High-Performance Liquid Chromatography⁻Tandem Mass Spectrometric Method for Determination of Folic Acid in Human Plasma.

There are health concerns associated with increased folic acid intake from fortified food and supplements. Existing analytical methods, however, which can be employed to carry out epidemiological and bioavailability studies for folic acid involve laborious sample preparation and/or lengthy chromatographic analysis. In this paper we describe a simple, rapid, and sensitive high-performance liquid chromatography–electrospray ionisation-tandem mass spectrometry (HPLC–ESI-MS/MS) method for determination of unmetabolised folic acid in human plasma using folic acid-d4 as an internal standard. The method required only a simple sample preparation step of protein precipitation and had a total run time of 3.5 min, which is the shortest run time reported to date for HPLC–MS/MS method employed for quantifying folic acid in plasma. The analytes were separated on a C18 column (3 µm; 50 × 3.00 mm) using an isocratic mobile phase consisting of ammonium acetate (1 mM)-acetic acid-acetonitrile (9.9:0.1:90, v/v/v). The method was fully validated in terms of accuracy, precision, linearity, selectivity, recovery, matrix effect, and stability. The short run time and the minimal sample preparation makes the method a valuable tool for performing high-throughput analyses. To demonstrate the applicability of the method in real conditions, it was applied successfully in a bioavailability study for the determination of unmetabolised folic acid levels in vivo in human plasma after oral administration of folic acid.

Validated Ultra Performance Liquid Chromatography-Tandem Mass Spectrometric Method for Determination of Betamethasone or Dexamethasone in Pharmaceuticals

Introduction: Chromatographic methods for determination of betamethasone or dexamethasone in their pharmaceutical preparations by Ultra Performance Liquid Chromatography-Tandem Mass Spectrometry were developed. Method: Analytes were eluted from Acquity BEH C18 (50 mm×2.1 mm, 1.7 µm) column using stepwise gradient with mobile phase consisted of acetonitrile and 0.1% (v/v) acetic acid in water. Quantitation of dexamethasone and betamethasone was performed using 451>361 m/z on MRM mode. Results: Methods were linear over the concentration range from 10 to 1500 ng mL -1 for both analyte. Limits of detection for BTM or DXM were 1 ng mL -1. The highest relative error and relative standard deviation values were not over 2%. Conclusion: Presented methods were specific, rugged and robust, successfully applied to the analysis of their pharmaceutical preparations. Keywords: Dexamethasone, betamethasone, UPLC-MS/MS, tablet, ampoule, drops.

Development and validation of a liquid chromatography/tandem mass spectrometric method for determination of ethinyl estradiol in human plasma

Introduction: A simple and rapid bioanalytical liquid chromatography-tandem mass spectrometry (LC-MS/MS) method based on solid phase extraction (SPE) followed by liquid-liquid extraction (LLE) has been developed and validated for quantification of Ethinyl Estradiol in human plasma. Methods: API 5500 LC-MS/MS system with turbo ion-spray interface equipped with pumps (Shimadzu LC-20ADVP), an auto Sampler (Shimadzu SIL-HTC), analytical column SB C18 HT (50 x 3.0 mm, 1.8 μ) and data acquisition system and quantitation program (Applied Biosystems Analyst Software version 1.5, Thermo scientific) was used. The Positive ions were measured in MRM mode for the analyte and Ethinyl Estradiol-d4 used as an internal standard. A composition of 2 mM ammonium formate buffer: acetonitrile (20: 80 v/v) was used as mobile phase. The total run time was 4.0 min. The proposed method has been validated with in the linear range of 5.000–308.560 pg/ml for Ethinyl Estradiol. Results: The retention times of Ethinyl Estradiol and Ethinyl estradiol-d4 were 3.42 min ± 0.30 min and 3.45 min ± 0.30 min respectively. The intraday and interday precision values were within 1.58% to 10.86% and 4.62% to 19.74% respectively for Ethinyl estradiol. The overall recovery for Ethinyl estradiol was found to be 86.91%-103.15%. Conclusion: The method was validated as per ICH guidelines and it would be useful for bioequivalence and pharmacokinetic studies of Ethinyl Estradiol in human plasma.

Validation and application of a liquid chromatography–electrospray ionization mass spectrometric method for determination of mazindol in human plasma and urine

IntroductionEven after removal of some stimulants, like fenproporex, amfepramone and mazindol, from Brazilian market, the use of these substances is still high, especially by drivers. Mazindol is the second most used anorectic agent in the world acting as an indirect sympathomimetic agonist, having stimulatory action on central nervous system. Plasma is a good matrix to monitor since it reflects the psychomotor effects of these drugs, but unlike urine has an invasive collection; drug levels and detection time are quite low. MethodThe method involved a liquid–liquid extraction of the samples and a LC–MS analysis was fully validated. Method was used to analyze samples of urine and plasma collected from health volunteers in a period of 24h. Metabolite of mazindol was synthesized using alkaline conditions. ResultsAfter validation the method proved to be adequate to analyze samples collected from health volunteers. Method was linear in the concentration range of 0.1–10ng/mL (r=0.9982) for plasma and 5–50ng/mL (r=0.9973) for urine. DiscussionAnalysis of the samples showed that mazindol can be detected after 1h of administration and that concentration levels in urine were always higher than in plasma. Mazindol metabolite was detected only in urine.

Development of a LC-MS-MS Method for Quantification of Valtrate and Its Application to Pharmacokinetic Study.

A simple and selective liquid chromatography-tandem mass spectrometric method for determination of valtrate in rat plasma was developed in this study. Chromatographic separation was achieved on a Thermo BDS HYPERSIL C18 column using acetonitrile-water-formic acid (75 : 25 : 0.1, v/v/v) as mobile phase in an isocratic mode of elution at a flow rate of 300 µL/min. MS-MS detection was performed in a positive ion electrospray ionization mode with the ion transitions 445.2 → 219.2 for valtrate and 355.2 → 135.1 for internal standard (sesamin). The developed method exhibited a linear dynamic range over 5.65-1695 ng/mL for valtrate in rat plasma. The overall extraction recovery of valtrate from plasma was 86.13-88.32%. The intra- and inter-day accuracy and precision were within the pre-defined limits of ≤15% at all concentrations. The method was successfully applied to pharmacokinetic studies of valtrate in rats.

Ultra performance liquid chromatography atmospheric pressure photoionization high resolution mass spectrometric method for determination of multiclass pesticide residues in grape and mango juices

A novel analytical method was developed for determination of organochlorine, synthetic pyrethroid, organophosphate and carbamate pesticide residues in fruit juices using ultra performance liquid chromatography–atmospheric pressure photoionization–high resolution mass spectrometry (UPLC–APPI–HRMS). The analytes were extracted from fruit juices by dispersive solid-phase extraction using multi-walled carbon nanotubes (MWCNTs). The analysis was carried out in full scan mode using dual ionization mode of APPI in the mass range of 100–650 units. The limit of detection and limit of quantification values for the pesticides were in the range of 0.025–0.15ngmL−1 and 0.1–0.5ngmL−1 respectively. The matrix effect of the method was found to be low and extraction recoveries were in the range of 60–110%. Some of the real fruits juice samples showed the presence of some pesticides in the range of 6.5–24.8ngL−1.

A rapid and sensitive liquid chromatography-tandem mass spectrometric method for determination of actinoside E in rat plasma and application to a pharmacokinetic study

A rapid and sensitive liquid chromatography-tandem mass spectrometric method for determination of actinoside E in rat plasma and application to a pharmacokinetic study

A rapid and sensitive liquid chromatography-tandem mass spectrometric method for determination of actinoside E in rat plasma and application to a pharmacokinetic study

A highly sensitive liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed for the determination of actinoside E in rat plasma. The analytes were extracted by ethyl acetate and an analogue of actinoside F was used as the internal standard. The mobile phase consisted of methanol-water (50: 50, V/V) containing 0.1% formic acid was delivered at a flow rate of 0.3 mL·min−1 to a Zorbax SB-C18 column (100 mm × 2.1 mm, 3.5 μm). The detection was performed by electrospray ionization mass spectrometry in the negative multiple reaction monitoring mode with a chromatograph run time of 3.0 min. Calibration curves of actinoside E were linear in the range of 0.5-2 500 ng·mL−1. In this range, intra- and inter-day precision ranged from 1.7% to 7.5% and 2.0% to 8.9%, respectively. The accuracy ranged from 95.7% to 108.6%, and extraction recovery from 83.2% to 85.5%. This method was successfully applied to a pharmacokinetic study of actinoside E in rats after intravenous (5 mg·kg−1) and oral (100 mg·kg−1) administration, and the results showed that actinoside E was poorly absorbed with an absolute bioavailability being approximately 0.27%.

LC–MS/MS determination of pramipexole on rat dried blood spots: A pharmacokinetic study

A simple and selective liquid chromatography-tandem mass spectrometric method for determination of pramipexole on rat dried blood spots was developed and validated. Chromatographic separation was achieved on a Synergy polar-RP column using 10mM ammonium acetate and methanol (50:50, v/v) as mobile phase in an isocratic mode of elution at a flow rate of 1.0mL/min. LC-MS was performed in a positive ion electro spray ionization mode and the MS/MS ion transitions 212.10→153.03 for PRX and 198.10→153.03 for internal standard (2-amino-4,5,6,7-tetrahydro-6-ethyl-amino-benzthiazole) were monitored. The developed method exhibited a linear dynamic range over 100-5000pg/mL for PRX on dried blood spots. The overall extraction recovery of PRX from DBS was 96.7%. The intra- and inter-day accuracy and precision were within the pre-defined limits of ≤15% at all concentrations. Influence of hematocrit and spot volume on dried blood spot was also evaluated and found to be well within the acceptable limits. The method was successfully applied to pharmacokinetic studies of PRX in rats.

Simultaneous Determination of 15 Plant Growth Regulators in Bean Sprout and Tomato with Liquid Chromatography–Triple Quadrupole Tandem Mass Spectrometry

The present paper describes the development and validation of a new reversed-phase liquid chromatography–electrospray ionization tandem mass spectrometric method for determination of 15 plant growth regulators in bean sprout and tomato. Plant growth regulators were extracted with mixed solvents methanol/acetonitrile/water/acetic acid (v/v/v/v, 40/40/20/1) from 10 g vegetable and analyzed directly without further cleanup. The identification of each analyte was established by chromatographic retention time, analyte-specific fragmentation patterns, and relative peak area ratios of precursor/product ion pairs. The recoveries of 15 plant growth regulators in bean sprout and tomato were 70.3–110.0 %. The repeatability of this method is excellent, and relative standard deviation value is less than 10 %.

Development and Validation of a Liquid Chromatographic/Tandem Mass Spectrometric Method for Determination of Chlortetracycline, Oxytetracycline, Tetracycline, and Doxycycline in Animal Feeds

A selective and accurate LC/MS/MS method for the simultaneous determination of chlortetracycline (CTC), oxytetracycline (OTC), tetracycline (TC), and doxycycline (DC) in animal feeds was developed. Samples were extracted with Na2EDTA-McIlvaine buffer and further purified with Oasis HLB SPE columns. The purified extract was separated on an Xbridge C18 column and detected by LC/MS/MS with positive electrospray ionization in the multiple reaction monitoring mode. This method provided average recoveries of 80.9 to 119.5%, with CVs of 1.7 to 9.8% in the range of 0.5 to 50 mg/kg CTC, OTC, TC, and DC in feeds, except the average recovery of CTC was 76.0%, with a CV of 14.6% in pig feed spiked with 0.5 mg/kg CTC. The linear ranges for the four TCs determined by LC/MS/MS ranged from 0.005 to 2.5 microg/mL with a linear correlation coefficient (R2) >0.99. The LOD and LOQ for CTC, OTC, TC, and DC in pig and poultry feeds ranged from 0.003 to 0.02 and 0.01 to 0.05 microg/g, respectively. The method was successfully applied for the analysis of 30 real feed samples, and no illegal use was detected.

Validation of a sequential extraction and liquid chromatography–tandem mass spectrometric method for determination of dihydrotestosterone, androstanediol and androstanediol–glucuronide in prostate tissues

Androgens are key mediators of prostate development and function, a role that extends to the development of prostate diseases such as benign prostatic hyperplasia (BPH) and prostate cancer. In prostate, DHT is the major androgen and reduction and glucuronidation are the major metabolic pathways for DHT elimination. A streamlined method for quantitation of dihydrotestosterone (DHT), 5α-androstan-3α,17β-diol (3α-diol), and 3α-diol glucuronide (diol-gluc) was established and validated for use with archived prostate tissue specimens to facilitate examination of the roles of the underlying metabolism. This involved a sequential 70/30 hexane/ethyl acetate (hex/EtOAc) extraction of steroids, followed by an ethyl acetate extraction for diol-gluc. Derivatization of the hex/EtOAc fraction with2-fluoro-1-methylpyridinium p-toluene-4-sulfonate (FMP) was used to enhance sensitivity for hydroxyl steroids and liquid chromatography–tandem mass spectrometry (LC–MS/MS) was utilized for analysis of both fractions. The method was validated with calibration standards followed by recovery assessment from spiked samples of BPH and normal prostate. Lower limits of quantitation (LLOQ) were 50pg/g, 20pg/g and 100pg/g for DHT, 3α-diol and diol-gluc, respectively for extracts from 50mg equivalents of tissue. Prepared samples were stable for up to three weeks at 4°C and 37°C. The method provides excellent sensitivity and selectivity for determination of tissue levels of DHT, 3α-diol, and diol-gluc. Furthermore, this protocol can easily be extended to other hydroxyl steroids, is relatively straightforward to perform and is an effective tool for assessing steroid levels in archived clinical prostate samples.

Development and validation of a liquid chromatographic-tandem mass spectrometric method for determination of eleven coccidiostats in milk

A reversed phase liquid chromatographic–tandem mass spectrometric method with simple solvent extraction and purification by solid phase extraction (SPE) has been developed for the determination of coccidiostats in milk. For sample preparation matrix solid phase dispersion, extraction by organic solvent and SPE with different cartridges were also tested. The compounds determined include lasalocid, narasin, salinomycin, monensin, semduramicin, maduramicin, robenidine, decoquinate, halofuginone, nicarbazin and diclazuril. Main steps of the method are addition of acetonitrile to the milk samples, centrifugation, removal of matrix by SPE, concentration by evaporation and LC–MS–MS determination. During a 15min time segmented chromatographic run compounds are ionised either positively or negatively. Calculated recoveries range between 77.1% and 118.2%. Maximum levels are in the range of 1–20μg/kg. The developed method was validated in line with the requirements of Commission Decision 2002/657/EC (2002). It is applicable for control of coccidiostat residues in milk as indicated in Regulation 124/2009/EC (2009).

Oral and topical pharmacokinetic studies of a novel TRPV1 antagonist, PAC-14028 in rats and minipigs using liquid chromatography/tandem mass spectrometric method

PAC-14028 ((E)-N-((R)-1-(3,5-difluoro-4-methanesulfonylamino-phenyl)-ethyl)-3-(2-propyl-6-trifluoromethyl-pyridine-3-yl)-acrylamide) is a novel and potent transient receptor potential vanilloid type I (TRPV1) antagonist. We developed and validated a rapid, sensitive and selective liquid chromatography/tandem mass spectrometric method for determination of PAC-14028 in rat and minipig plasma. After protein precipitation PAC-14028 and internal standard (methylated analog, PAC-14026) were separated on a Symmetry C 18 column (4.6 mm × 75 mm, 3.5 μm) with an isocratic mobile phase, acetonitrile: water (8:2, v/v) containing 0.2% formic acid and monitored by electrospray positive ionization with multiple reaction monitoring mode (PAC-14028, 492 → 156; IS, 506 → 156, m/ z). The calibration curve was linear over the range of 1.0–500 ng/ml ( r 2 > 0.999) and lower limit of quantitation (LLOQ) was 1 ng/ml. The precision and accuracy were within ±15% and the stability was acceptable during bench-top, auto-sampler, 3 freeze–thaw cycles and 4-week storage in a freezer at −80 °C. This method was successfully applied to the intravenous, oral and topical pharmacokinetic studies of PAC-14028 in rats and minipigs, which showed comparable pharmacokinetic parameters (T1/2, 2.1 h and 3.8 h; F%, 52.7% and 64.2% for rats and minipigs, respectively). Percutaneous absorption of PAC-14028 was negligible after topical application ( F% 0.2–1.7%).

Method for determining the tritium concentration using the effects of dissociation of molecular beams of hydrogen isotopes on thin carbon films

Physical foundations of a new method for mass-spectrometric determination of tritium concentration in hydrogen-containing media, which is based on the effect of dissociation of molecular ions of hydrogen isotopes passing through thin carbon films, are formulated. The effects accompanying the interaction of particles with a solid (angular scattering, change in the charge state, energy losses in protons, deuterons, and tritons) are analyzed. The corresponding theoretical and experimental data indicate that the proposed technology can be implemented in practice for a comparatively low particle energy of ∼10 keV/nucleus.