Quantitative determination of cationic modified polysaccharides on hair using LC–MS and LC–MS–MS

Cationic polysaccharides containing N-hydroxypropyl-N,N,N-trimethylammonium substituents are widely used as conditioning agents for hair-care products. A sensitive method has been developed for the quantitation of these polymers. After acidic extraction from hair the polysaccharides are hydrolyzed using trifluoroacetic acid. The cationic monoglycosides are determined using liquid chromatography–tandem mass spectrometry (LC–MS–MS). The developed method is independent of hair treatment. Even hair cut from test persons after customary hair wash can be analyzed. After treatment of natural and bleached hair tresses using a real-life treatment procedure 180 g and 300 g of polymer per gram hair were quantified, respectively. Additionally the fragmentation mechanism of the cationic N-hydroxypropyl-N,N,N-trimethylammonium group during electrospray ionization was investigated. A mass loss of 60 Da in combination with loss of a single charge is observed and associated with cleavage of trimethylamine and a proton. It is assumed that this process is promoted by the anionic counter-ion which might be hydroxide in an aqueous environment.     

LC–MS–MS Determination of Rotenone, Deguelin, and Rotenolone in Human Serum

For the first time we report a rapid and sensitive LC–MS–MS method for quantification of rotenone, deguelin, and rotenolone in human serum. The analytical procedure involves extraction with ethyl acetate without further clean-up. The active ingredients were separated on a C₈ reversed-phase column by isocratic elution. Eleven simultaneous transitions of precursor ions were monitored. Excellent selectivity and sensitivity enables quantification and identification of low levels of rotenoids (LOD 2 ng mL^?1, LOQ 5 ng mL^?1) in human serum.     

Stress Studies and Identification of Degradation Products of Cephalexin Using LC–PDA and LC–MS/MS

A stability indicating RP-HPLC method for cephalexin has been developed and validated to identify and characterize potential degradation products. Drug was subjected to hydrolytic (acidic, basic, and neutral), oxidative, thermal, and photolytic stresses as per ICH guidelines Q1A (R2) and Q1B. Chromatographic separation was achieved on C8 column with mixture of ammonium acetate buffer pH 4.5 and acetonitrile in gradient mode as a mobile phase with PDA detection. Specificity of the method was established by peak purity studies. Method was validated as per ICH guideline Q2 (R1) for accuracy, precision, linearity, sensitivity, and robustness. Kinetics for each degradation condition was studied with respect to order of reaction and rate constant. Method was found to comply with acceptance criteria of validation parameters with respect to specificity (peak purity greater than 0.999) linearity (r ² greater than 0.99), accuracy (% recovery in the range of 98–102%), and precision (% RSD not more than 2). A total of six degradation products were generated in different stress conditions; these were identified and structures were proposed using LC–MS/MS. Cephalexin undergoes degradation in almost all the conditions. The developed stability indicating method is suitable for analysis of stability samples as it adequately separates all degradation products. Degradation products generated in photolytic and oxidative conditions are reported for the first time.     

Comparison of LC–UV, LC–ELSD and LC–MS Methods for the Determination of Sesquiterpenoids in Various Species of Artemisia

Simple and specific analytical methods for the quantitative determination of sesquiterpenoids from various species of Artemisia plant samples were developed. By LC–UV, LC–ELSD, the separation was achieved by reversed-phase chromatography on a C18 column with water and acetonitrile both containing 0.025% trifluoroacetic acid as the mobile phase. In the LC–MS system, trifluoroacetic acid was replaced by 0.1% formic acid. The wavelength used for quantification of sesquiterpenoids with a diode array detector was 205 nm. The limits of detection by LC–MS was found to be 5, 10, 25, 50, 50 ng mL^?1. The limits of detection by LC–UV and LC–ELSD were found to be 5.0, 3.0, 100, 100, 7.5 μg mL^?1, by LC–UV and 50, 25, 30, 100 and 75 μg mL^?1 by LC–ELSD. LC–mass spectrometry coupled with electrospray ionization (ESI) interface is described for the identification and quantification of sesquiterpenoids in various plant samples. This method involved the use of the [M + H]⁺ ions of sesquiterpenoids in the positive ion mode with extractive ion monitoring.     

Investigation of Euphrasia rostkoviana Hayne Using GC–MS and LC–MS

Gas and liquid chromatography coupled with mass spectrometry was applied to solve difficulties and reinvestigate the serious matrix problems affecting analysis of the active compounds in Euphrasia rostkoviana Hayne. The main groups of compounds were obtained by extracting the herb stepwise with n-hexane, chloroform, ethyl acetate and methanol. Polyamide column chromatography facilitated further separation. Phenolic/flavonoid- and terpenoid-type molecules were studied by GC–MS, HPLC and LC–MS–MS. The β-sitosterol content of the herb was determined by gas chromatography with flame ionisation detection (GC-FID). Caffeic acid, chlorogenic acid, coumaric acid and flavonoid glycosides of apigenin, luteolin, rhamnetine (hexoside), kaempferol (both hexoside and rutinoside) and quercetin (rutinoside) were identified in the fractions of the methanolic extract.     

LC–MS–MS Determination of Stabilizers and Explosives Residues in Hand-Swabs

The contribution of explosive trace detection in samples from the hands of suspects has been fundamental in several forensic cases involving terrorists. This paper describes a method for the rapid extraction and unequivocal confirmation of some high potential explosives (trinitrotoluene, cyclotrimethylenetrinitramine, pentaerythritol tetranitrate, nitroglycerin) and two stabilizer (diphenylamine and ethylcentralite) residues in hand-swabs using liquid chromatography–tandem mass spectrometry. The extraction procedure of the analytes from the swabs is realized by solvent elution and the extracts are directly analyzed. Recoveries from spiked swabs range from 78 to 96%; the limits of quantification are between 0.04 and 1.8 ng injected and the inter-day method precision is less than 15%. The developed procedure was applied to the detection of explosives traces in samples after handling tests.     

LC–MS–MS Analysis of Mirtazapine in Plasma, and Determination of Pharmacokinetic Data for Rats

A sensitive LC–MS–MS method with electrospray ionization has been developed for analysis of mirtazapine in rat plasma. After addition of diazepam as internal standard, liquid–liquid extraction was used to produce a protein-free extract. Chromatographic separation was achieved on a 150 × 4.6 mm, 5 μm particle, ODS column with 84:16 (v/v) methanol–water containing 0.1% ammonium acetate and 0.01% glacial acetic acid as mobile phase. LC–MS–MS was performed in selected-ion-monitoring (SIM) mode using target fragment ions m/z 195.09 for mirtazapine and m/z 192.80 for the IS. Calibration plots were linear over the range of 0.516–618.8 ng mL^?1. The lower limit of quantification was 0.516 ng mL^?1. Intra-day and inter-day precision were better than 12.6 and 8.8%, respectively. Mean recovery of mirtazapine from plasma was in the range 87.41–90.06%; average recovery was 88.40% (RSD 3.95%). Significant gender differences between mirtazapine pharmacokinetic data were observed in this study.     

Determination of Fenofibric Acid in Human Plasma by LC–MS/MS and LC–UV

A sensitive, high-throughput and economic liquid chromatographic method for determination of fenofibric acid in human plasma was developed and validated by ultraviolet detection and tandem mass spectrometry, then applied in pharmacokinetic study to investigate Lipanthyl™ 200 mg MC bioavailability under food and fasting conditions. Fenofibric acid with 2-chloro fenofibric acid-d6 (internal standard) was extracted from 100 µL of human plasma by acetonitrile in a single extraction step. 25 and 2 µL from supernatant were injected onto ACE column, 50 mm, 5 micron with 4.6 mm inner diameter for LC–UV and 2.1 mm for LC–MS/MS, and both systems were eluted isocratically by water:methanol:formic acid (35:65:0.1, v/v/v), with a constant flow rate of 1 mL min⁻¹. The established calibration curve was linear between 0.05–20 µg mL⁻¹, and the within- and between-day precisions were all below 13 % in both LC–MS/MS and LC–UV systems during validation, and accuracies ranged between 91 and 112 %. Twenty-eight healthy adult subjects participated in this clinical study, and the pharmacokinetic parameters including coefficient of variation were calculated and discussed. A dramatic decrease in C _max and AUC0-72 (3.63- and 1.85-fold, respectively) were observed for Lipanthyl™ MC under fasting conditions with more variable inter subject measurements comparing to the fed state.

     

Detection and Confirmation of Ginsenosides in Horse Urine by GC–MS and LC–MS

Ginseng has been used by the Chinese as a traditional herbal medicine for thousands of years. In view of the growing popularity in the use of ginseng preparations as natural remedies and food supplements worldwide, there is an increasing concern for their abuse in both human and animal sports. Ginsenosides are considered the major constituents of ginseng responsible for its pharmacological properties. In this study, a method was developed for the detection and confirmation of a number of ginsenosides in horse urine. The intact ginsenosides were detected and confirmed at 5–100 ng mL^?1 by LC–MS², and two deglycosylation metabolites, namely protopanaxadiol and protopanaxatriol, could both be detected and confirmed at 2 ng mL^?1 by GC–MS² after trimethylsilylation. The above GC–MS and LC–MS methods were then applied to study the in vitro metabolism of ginsenosides Rg1 and Rb1 and the in vivo urinary metabolites after oral administration of Rg1 to horses. Results obtained reveal the very first evidence for the existence of the metabolites, Rg1 and protopanaxatriol, as glucuronides in urine.     

Qualitative metabolism assessment and toxicological detection of xylazine, a veterinary tranquilizer and drug of abuse, in rat and human urine using GC–MS, LC–MS n , and LC–HR-MS n

Xylazine is used in veterinary medicine for sedation, anesthesia, and analgesia. It has also been reported to be misused as a horse doping agent, a drug of abuse, a drug for attempted sexual assault, and as source of accidental or intended poisonings. So far, no data concerning human metabolism have been described. Such data are necessary for the development of toxicological detection methods for monitoring drug abuse, as in most cases the metabolites are the analytical targets. Therefore, the metabolism of xylazine was investigated in rat and human urine after several sample workup procedures. The metabolites were identified using gas chromatography (GC)–mass spectrometry (MS) and liquid chromatography (LC) coupled with linear ion trap high-resolution multistage MS (MS ⁿ ). Xylazine was N-dealkylated and S-dealkylated, oxidized, and/or hydroxylated to 12 phase I metabolites. The phenolic metabolites were partly excreted as glucuronides or sulfates. All phase I and phase II metabolites identified in rat urine were also detected in human urine. In rat urine after a low dose as well as in human urine after an overdose, mainly the hydroxy metabolites were detected using the authors’ standard urine screening approaches by GC–MS and LC–MS ⁿ . Thus, it should be possible to monitor application of xylazine assuming similar toxicokinetics in humans.

Advantages of LC–MS–MS compared to LC–MS for the determination of nitrofuran residues in honey

In the framework of developing analyses for exogenous contaminants in food matrices such as honey, we have compared data obtained by high-performance liquid chromatography coupled with mass spectrometry (LC–MS) to those provided by high-performance liquid chromatography and tandem mass spectrometry (LC–MS–MS). Initial results obtained with LC–MS showed that the technique lacked selectivity, which is why the method was validated by LC–MS–MS. This method involves a solid-phase extraction (SPE) of nitrofuran metabolites and nitrofuran parent drugs, a derivatization by 2-nitrobenzaldehyde for 17 h, and finally a clean-up by SPE. The data obtained show that the limits of detection varied between 0.2 and 0.6 μg kg⁻¹ for the metabolites and between 1 and 2 μg kg⁻¹ for nitrofuran parent drugs. The method was applied to different flower honeys. The results showed that nitrofurans (used as antibiotics) are consistently present in this matrix, the predominant compound being furazolidone. Figure Working bees     

LC–MS/MS and NMR characterization of forced degradation products of mirabegron

A rapid, precise, and reliable liquid chromatography tandem mass spectrometry (LC–MS/MS) method has been developed for the characterization of stressed degradation products of mirabegron. It is used in the treatment of overactive bladder and administered to treat urinary symptoms such as urgency or frequency and incontinence. It also works by relaxing the muscles around bladder.

Mirabegron was subjected to hydrolysis (acidic, alkaline, and neutral) and peroxidation, as per ICH-specified conditions. The drug showed degradation under stress conditions. However, it was stable to neutral conditions. A total of seven degradation products were observed and the chromatographic separation of the drug and its degradation products was achieved on X-TerraRP-8 (250 mm × 4.6 mm, i.d., 5 µm) column using 0.01 M ammonium acetate as mobile phase-A and 60:40 ratio of acetonitrile (ACN):water as mobile phase-B. The degradation products were characterized by LC–MS/MS and its fragmentation pathways were proposed. Probable possible structures were drawn based on parent and daughter molecular ions. One peroxide degradant impurity was isolated using preparative LC and characterized using liquid chromatography–mass spectrometry and NMR data.     

Simultaneous Determination of Levodopa, Benserazide and 3-O-Methyldopa in Human Serum by LC–MS–MS

For the first time a sensitive, specific and rapid LC–MS–MS assay is presented for the simultaneous determination of levodopa (L-DP), 3-O-methyldopa (3-OMD) and benserazide (BSZ) in human serum. The three compounds were extracted from human serum by protein precipitation followed by dilution of the supernatant with aqueous formic acid. In serum, linearity was observed between 50 and 1,000 ng mL^?1 of L-DP, 3-OMD and BSZ, respectively. Intra-day and inter-day RSD values were below 10.56 and 6.22% at concentrations of 120, 360 and 720 ng mL^?1. The presented method showed excellent specificity and sensitivity compared with other methods reported. It was applied to a pharmacokinetic study and demonstrated its applicability to pre-clinical and clinical pharmacological research.     

Assessment of robustness for an LC–MS–MS multi-method by response-surface methodology,and its sensitivity

Sensitive, fast, and robust multi-methods are required for the surveillance of the contamination of the drinking water resources by organic trace contaminants. In the present work an alternative strategy using response surface methodology (RSM) was applied for assessment of the robustness of a LC–MS–MS multi-method. The analytical method was optimised by means of a central composite design including six design variables. The main object was to evaluate the significance of the RSM results with regard to robustness and to the sensitivity to the mass transitions used in the multi-method. The robustness of the multi-method was represented by the curvature of the calculated response surfaces for the response value R. Furthermore, it could be demonstrated that the RSM was sensitive to changes made to the investigated data set and was able to clearly indicate the fraction of substances, which met the defined criterion for signal-to-noise-ratio.     

LC–DAD and LC–ESI–MS Chromatographic Fingerprinting and Quantitative Analysis for Evaluation of the Quality of Huang-Lian-Jie-Du-Tang

LC–DAD coupled with electrospray tandem mass spectrometry (LC–ESI–MS–MS) has been used to evaluate the quality of the traditional Chinese medicine Huang-Lian-Jie-Du-Tang (HLJDT). Twenty-five chromatographic peaks were obtained from a C₁₈ analytical column by gradient elution with acetonitrile and formate buffer (containing 0.5% formic acid) at a flow rate of 1.0 mL min^?1. The linearity, precision, and accuracy of the data obtained were acceptable. Thirteen components were identified by ESI–MS, and seven of these were quantitatively analyzed by LC–DAD. The method was used to analyze ten batches of HLJDT, and both chromatographic fingerprints and quantitative data were used to evaluate the quality of the HLJDT. It was concluded that this LC–DAD–ESI–MS method enables more fully validated and complete evaluation and monitoring of the quality of HLJDT.     

Evaluation of Sempervivum tectorum L. Flavonoids by LC and LC–MS

A reversed-phase high-performance liquid chromatography method with UV detection was developed for determination of [(N-morpholine)methylene]daunorubicin hydrochloride (DD-M) during studies of its stability. In this LC method the following were used: an RP-column, the mobile phase—acetonitrile:methanol:solution A (9:1:10 v/v/v) [solution A contains 2.88 g of sodium laurilsulfate and 1.6 mL of phosphoric acid(V)] with a flow rate of 1.4 mol L⁻¹ and quinine hydrochloride as an internal standard. The detection wavelength was 254 nm. The method was validated with regard to linearity, limit of detection, limit of quantitation, selectivity and precision. Hydrolysis of the DD-M catalyzed by hydrogen ions in hydrochloric acid and a spontaneous reaction of the DD-M degradation under the influence of the water in sodium hydroxide took place. The thermodynamic parameters of these reactions—energy, enthalpy and entropy of activation—were calculated. It was observed that a positive salt effect occurred in hydrochloric acid.

     

Simultaneous Determination of Tamsulosin and Dutasteride in Human Plasma by LC–MS–MS

A sensitive and selective liquid chromatographic tandem mass spectrometric (LC–MS–MS) method was developed for simultaneous identification and quantification of tamsulosin and dutasteride in human plasma, which was well applied to clinical study. The method was based on liquid–liquid extraction, followed by an LC procedure with a Gemini C-18, 50 mm × 2.0 mm (3 μm) column and using methanol:ammonium formate (97:3, v/v) as the mobile phase. Protonated ions formed by a turbo ionspray in positive mode were used to detect analytes and internal standard. MS–MS detection was by monitoring the fragmentation of 409.1 → 228.1 (m/z) for tamsulosin, 529.3 → 461.3 (m/z) for dutasteride and 373.2 → 305.3 (m/z) for finasteride (IS) on a triple quadrupole mass spectrometer. The lower limit of quantification for both tamsulosin and dutasteride was 1 ng mL^?1. The proposed method enables the unambiguous identification and quantification of tamsulosin and dutasteride for clinical drug monitoring.     

Multiresidue Analysis of Pesticides in Vegetables and Citrus Fruits by LC–MS–MS

In this work, an analytical multiresidue method using liquid chromatography tandem-mass spectrometry (LC–MS–MS) with triple quadrupole in selected reaction monitoring (SRM) mode for the simultaneous determination of 54 pesticides in vegetables (pepper and tomato) and citrus fruits (orange and lemon) has been developed. The procedure involves initial single phase extraction of sample with acetonitrile by agitation, followed by liquid–liquid partition aided by “salting out” process using NaCl. The average recovery by the LC–MS–MS method obtained for these compounds varied from 65.5 to 114.5% with a relative standard deviation between 2.3 and 8.3%. The method presents good linearity over the range assayed 10–500 μg L^?1 (except famoxadone 50–1,000 μg L^?1) and the detection limits for the pesticides studied varied from 0.03 to 14.9 μg kg^?1. The proposed method was used to determine pesticide levels in vegetables and citrus fruit samples from different experimental orchards and greenhouses from the Region of Murcia.     

Simultaneous Determination of Atenolol and Chlorthalidone by LC–MS–MS in Human Plasma

A simple, sensitive, selective and rapid liquid chromatography–tandem mass spectrometry method was developed and validated for the simultaneous separation and quantitation of atenolol and chlorthalidone in human plasma using metoprolol and hydrochlorothiazide as internal standard. Following solid phase extraction, the analytes were separated by an isocratic mobile phase on a reversed-phase C₁₈ column and analyzed by MS in the multiple reaction-monitoring mode (atenolol in positive and chlorthalidone in the negative ion mode). The limit of quantitation for this method was 10 and 15 ng mL^?1 and the linear dynamic range was generally 10–2,050 ng mL^?1 and 15–3,035 ng mL^?1 for atenolol and chlorthalidone, respectively.     

LC–MS–MS Determination of Nikethamide in Human Plasma

A sensitive LC–MS–MS method with electrospray ionization has been developed for determination of nikethamide in human plasma. After addition of atropine as internal standard, liquid–liquid extraction was used to produce a protein-free extract. Chromatographic separation was achieved on a 150 mm × 2.1 mm, 5 μm particle, Agilent Zorbax SB-C₁₈ column, with 45:55 (v/v) methanol–water containing 0.1% formic acid as mobile phase. LC–MS–MS was performed in multiple reaction monitoring mode using target fragment ions m/z 178.8 → 107.8 for nikethamide and m/z 289.9 → 123.8 for the internal standard. Calibration plots were linear over the range of 20.0–2,000 ng mL^?1. The lower limit of quantification was 20.0 ng mL^?1. Intra-day and inter-day precisions were better than 4.2 and 6.1%, respectively. Mean recovery of nikethamide from human plasma was in the range 65.3–71.1%.