A review on development of analytical methods to determine monitorable drugs in serum and urine by micellar liquid chromatography using direct injection

Therapeutic drug monitoring is a common practice in clinical studies. It requires the quantification of drugs in biological fluids. Micellar liquid chromatography (MLC), a well-established branch of Reverse Phase-High Performance Liquid Chromatography (RP-HPLC), has been proven by many researchers as a useful tool for the analysis of these matrices. This review presents several analytical methods, taken from the literature, devoted to the determination of several monitorable drugs in serum and urine by micellar liquid chromatography. The studied groups are: anticonvulsants, antiarrhythmics, tricyclic antidepressants, selective serotonin reuptake inhibitors, analgesics and bronchodilators. We detail the optimization strategy of the sample preparation and the main chromatographic conditions, such as the type of column, mobile phase composition (surfactant, organic solvent and pH), and detection. The finally selected experimental parameters, the validation, and some applications have also been described. In addition, their performances and advantages have been discussed. The main ones were the possibility of direct injection, and the efficient chromatographic elution, in spite of the complexity of the biological fluids. For each substance, the measured concentrations were accurate and precise at their respective therapeutic range. It was found that the MLC-procedures are fast, simple, inexpensive, ecofriendly, safe, selective, enough sensitive and reliable. Therefore, they represent an excellent alternative for the determination of drugs in serum and urine for monitoring purposes.     

Development and validation of LC-HRMS and GC-NICI-MS methods for stereoselective determination of MDMA and its phase I and II metabolites in human urine

3,4-Methylenedioxymethamphetamine (MDMA) is a racemic drug of abuse and its R- and S-enantiomers are known to differ in their dose-response curve. The S-enantiomer was shown to be eliminated at a higher rate than the R-enantiomer most likely explained by stereoselective metabolism that was observed in various in vitro experiments. The aim of this work was the development and validation of methods for evaluating the stereoselective elimination of phase I and particularly phase II metabolites of MDMA in human urine. Urine samples were divided into three different methods. Method A allowed stereoselective determination of the 4-hydroxy-3-methoxymethamphetamine (HMMA) glucuronides and only achiral determination of the intact sulfate conjugates of HMMA and 3,4-dihydroxymethamphetamine (DHMA) after C18 solid-phase extraction by liquid chromatography-high-resolution mass spectrometry with electrospray ionization. Method B allowed the determination of the enantiomer ratios of DHMA and HMMA sulfate conjugates after selective enzymatic cleavage and chiral analysis of the corresponding deconjugated metabolites after chiral derivatization with S-heptafluorobutyrylprolyl chloride using gas chromatography-mass spectrometry with negative-ion chemical ionization. Method C allowed the chiral determination of MDMA and its unconjugated metabolites using method B without sulfate cleavage. The validation process including specificity, recovery, matrix effects, process efficiency, accuracy and precision, stabilities and limits of quantification and detection showed that all methods were selective, sensitive, accurate and precise for all tested analytes.     

Densitometry and indirect normal‐phase HPTLC–ESI–MS for separation and quantitation of drugs and their glucuronide metabolites from plasma

The present work describes novel methods using densitometry and indirect or off‐line high performance thin‐layer chromatography–mass spectrometry (HPTLC–MS) for the simultaneous detection and quantification of asenapine, propranolol and telmisartan and their phase II glucuronide metabolites. After chromatographic separation of the drugs and their metabolites the analytes were scraped, extracted in methanol and concentrated prior to mass spectrometric analysis. Different combinations of toluene and methanol–ethanol–n‐butanol–iso‐propanol were tested for analyte separation and the best results were obtained using toluene–methanol–ammonia (6.9:3.0:0.1, v/v/v) as the elution solvent. All of the drug–metabolite pairs were separated with a homologous retardation factor difference of ≥22. The conventional densitometric approach was also studied and the method performances were compared. Both of the approaches were validated following the International Conference on Harmonization guidelines, and applied to spiked human plasma samples. The major advantage of the TLC–MS approach is that it can provide much lower limits of detection (1.98–5.83 pg/band) and limit of quantitation (5.97–17.63 pg/band) with good precision (?3.0% coefficient of variation) compared with TLC–densitometry. The proposed indirect HPTLC–MS method is simple yet effective and has tremendous potential in the separation and quantitation of drugs and their metabolites from biological samples, especially for clinical studies.     

Development and validation of a quantification method for ziyuglycoside I and II in rat plasma: Application to their pharmacokinetic studies

This study provided a novel and generally applicable method to determine ziyuglycoside I and ziyuglycoside II in rat plasma based on liquid chromatography with tandem mass spectrometry. A single step of liquid–liquid extraction with n‐butanol was utilized, and ginsenoside Rg3 was chosen as internal standard. Final extracts were analyzed based on liquid chromatography with tandem mass spectrometry. Chromatographic separation was achieved using a Thermo Golden C₁₈ column, and the applied gradient elution program allowed for the simultaneous determination of two ziyuglycosides in a one‐step chromatographic separation with a total run time of 10 min. The fully validated methodology for both analytes demonstrated high sensitivity (the lower limit of quantitation was 2.0 ng/mL), good accuracy (% RE ≤ ± 15) and precision (% RSD ≤ 15). The average recoveries of both ziyuglycosides and internal standard were all above 75% and no obvious matrix effect was found. This method was then successfully applied to the preclinical pharmacokinetic studies of ziyuglycoside I and ziyuglycoside II. The presently developed methodology would be useful for the preclinical and clinical pharmacokinetic studies for ziyuglycoside I and ziyuglycoside II.     

Stability and degradation products of imipenem applying high‐resolution mass spectrometry: An analytical study focused on solutions for infusion

Carbapenems show recognized instability in aqueous solutions; therefore some care must be taken in their handling and preparation and their use in the hospital environment. The stability and degradation products of imipenem were investigated from conditions that simulate its clinical use. For this, a simple stability‐indicating method by HPLC‐DAD was validated with a focus on the quantitation of drug concentration remaining from infusion solutions (sodium chloride 0.9% and glucose 5%). The degradation products formed were identified by high‐resolution mass spectrometry (ESI‐Q‐TOF‐MS/MS), with detection of the [M + H]⁺ ions at m/z 318 (DP‐1), m/z 599 (DP‐2) and m/z 658 (DP‐3). The most probable elemental compositions were obtained with a high degree of confidence, where the error between the masses observed and calculated was 1.25 ppm for DP‐1, ?0.33 ppm for DP‐2 and 1.82 ppm for DP‐3. The DP‐1 degradation product resulted from cleavage of the β‐lactam ring; DP‐2 corresponded to the drug dimer; and DP‐3 was generated from the interaction between imipenem and cilastatin. The proposed method provides a safe and reliable alternative for the quantitation of imipenem, and the stability data obtained by ESI‐Q‐TOF help in understanding the drug behavior under the conditions of clinical use.     

Ultra‐rapid non‐equilibrium solid‐phase microextraction at elevated temperatures and direct coupling to mass spectrometry for the analysis of lidocaine in urine

Solid‐phase microextraction (SPME) has been directly coupled to an ion‐trap mass spectrometer (MS) for the determination of the model compound lidocaine in urine, hereby applying MS/MS [fragmentation of [M + H]⁺ (m/z 235) to a fragment with m/z 86]. The throughput of samples has been increased using non‐equilibrium SPME with polydimethylsiloxane (PDMS) fibers. The effect of temperature on the sorption and the desorption was studied. Elevated temperatures during sorption (65°C) and desorption (55°C) had a considerable influence on the speed of the extraction. The desorption was carried out with a home‐made desorption chamber allowing thermostating. Only 1 min sorption and 1 min desorption were performed, after which MS detection took place, resulting in a total analysis time of 3 min. Detection limits below 1 ng/mL could be obtained despite yields of only 2.1 and 1.5% for a 100‐ and a 30‐μm PDMS‐coated fiber, respectively. Furthermore, the determination of lidocaine in urine had acceptable reproducibilities, i.e., relative standard deviations (RSDs) below 10%. A limit of quantitation (RSD < 15%) of about 1 ng/mL was obtained. No extra wash step of the extraction fiber was required after desorption if a 30‐μm coating was used, whereas not all the analyte was desorbed from the 100‐μm coating in a single desorption. Therefore, the SPME‐MS/MS system with a 30‐μm PDMS‐coated fiber for rapid non‐equilibrium SPME at elevated temperatures has interesting potential for high‐throughput analysis of biological samples.     

Multi-residue analytical methods using LC-tandem MS for the determination of pharmaceuticals in environmental and wastewater samples: a review

Multi-residue analytical methodologies are becoming the preferred and required tools against single group analysis, as they provide wider knowledge about the occurrence of pharmaceuticals in the environment necessary for further study of their removal, partition and ultimate fate. However, simultaneous analysis of compounds from different groups with quite different physico-chemical characteristics requires a compromise in the selection of experimental conditions, which in some cases are not the best conditions for all the analytes studied. In this article, an overview of analytical methodologies focusing on the simultaneous determination of acidic, neutral and basic compounds belonging to different therapeutical classes is presented. The state-of-the-art of LC-MS/MS for multi-class analysis is reviewed, highlighting the specific requirements for such analysis.     

From large analogical instruments to small digital black boxes: 40 years of progress in mass spectrometry and its role in proteomics. Part II 1985–2000

This is the continuation of a personal retrospective on the developments that since 1965 have given shape to Mass Spectrometry (MS) and taken it from a position of simply playing a role in Protein Chemistry to becoming an indispensable tool in Proteomics, all within a 40‐year span. Part I covered the period from 1965 to 1984. This second part reviews the Mass Spectrometry timeline of events from 1985 to 2000, stopping at various time points where MS made significant contributions to protein chemistry or where the development of new instrumentation for MS represented a major advance for peptide and protein work. Major highlights in the field and their significance for peptide and protein characterization such as the advent and practical consequences of electrospray ionization (ESI) and matrix‐assisted laser desorption/ionization (MALDI) are covered, including work done with triple quads, the development of time‐of‐flight (TOF) instruments and new ion traps and going on to the more recent work on the full characterization of the Proteome with ion traps, TOF instruments and new ionization and tagging techniques for protein sequencing. Copyright © 2009 John Wiley & Sons, Ltd.     

Development of a multi-residue analytical methodology based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) for screening and trace level determination of pharmaceuticals in surface and wastewaters

This paper describes development, optimization and validation of a method for the simultaneous determination of 29 multi-class pharmaceuticals using off line solid phase extraction (SPE) followed by liquid chromatography-triple quadrupole mass spectrometry (LC-MS-MS). Target compounds include analgesics and non-steroidal anti-inflammatories (NSAIDs), lipid regulators, psychiatric drugs, anti-histaminics, anti-ulcer agent, antibiotics and β-blockers. Recoveries obtained were generally higher than 60% for both surface and wastewaters, with exception of several compounds that yielded lower, but still acceptable recoveries: ranitidine (50%), sotalol (50%), famotidine (50%) and mevastatin (34%). The overall variability of the method was below 15%, for all compounds and all tested matrices. Method detection limits (MDL) varied between 1 and 30 ng/L and from 3 to 160 ng/L for surface and wastewaters, respectively. The precision of the method, calculated as relative standard deviation (R.S.D.), ranged from 0.2 to 6% and from 1 to 11% for inter and intra-day analysis, respectively. A detailed study of matrix effects was performed in order to evaluate the suitability of different calibration approaches (matrix-matched external calibration, internal calibration, extract dilution) to reduce analyte suppression or enhancement during instrumental analysis. The main advantages and drawbacks of each approach are demonstrated, justifying the selection of internal standard calibration as the most suitable approach for our study. The developed analytical method was successfully applied to the analysis of pharmaceutical residues in WWTP influents and effluents, as well as in river water. For both, river and wastewaters, the most ubiquitous compounds belonged to the group of anti-inflammatories and analgesics, antibiotics, the lipid regulators being acetaminophen, trimethoprim, ibuprofen, ketoprofen, atenolol, propranolol, mevastatin, carbamazepine and ranitidine the most frequently detected compounds.     

Determination of seven drugs of abuse and their metabolites in surface and wastewater using solid‐phase extraction coupled to liquid chromatography with high‐resolution mass spectrometry

A method based on liquid chromatography with electrospray ionization high‐resolution mass spectrometry (Exactive Orbitrap) combined with solid‐phase extraction using a strong cationic exchange mixed‐mode sorbent has been developed for the determination of seven drugs of abuse, including two synthetic cathinones, as well as some of their metabolites in environmental water samples. The method provides low detection limits and a high confirmation power thanks to the diagnostic and two fragment ions monitored for each compound in high‐resolution mass spectrometry, providing six identification points for each analyte. The clean‐up step based on methanol in the extraction step adequately decreased the matrix effect, mainly for river and effluent water, and provided suitable process efficiency. Method detection and quantitation limits for environmental waters were at low nanogram per liter. The method was applied to analyze the samples of influent and effluent wastewater, as well as surface water. Codeine, methadone, and its metabolite were determined in all samples of wastewater and the metabolite of cocaine, benzoylecgonine, was found at the highest concentration.     

From large analogical instruments to small digital black boxes: 40 years of progress in mass spectrometry and its role in proteomics. Part I 1965-1984

As the title implies, the author undertakes a personal retrospective on the developments that since 1965 have shaped MS and taken it from a position of simply playing a role in protein chemistry to becoming an indispensable tool in proteomics, all in the past 40-year span. The article reviews the MS timeline of events, stopping at various time points where MS made significant contributions to protein chemistry or where the development of new instrumentation for MS represented a major advance for peptide and protein work. Major highlights in the field and their significance for peptide and protein characterization are covered, starting from the pioneering work carried out in the 1960s on peptide derivative formation and sequencing with instrumentation proper of that time, to later work done with triple, quad, and four-sector instruments, and on to the more recent work on the characterization of the proteome with ion traps, time-of-flight (TOF) instruments, and new ionization and tagging techniques.     

Evaluation of the limit of performance of an analytical method based on a statistical calculation of its critical concentrations according to ISO standard 11843: Application to routine control of banned veterinary drug residues in food according to European Decision 657/2002/EC

Traceability of the measurement of analytical parameters capable of evaluating the performance of methods is an important concept for the assessment of quality for the routine control, especially for residue monitoring of non-authorized medicinal substances in food from animal origin. The European Decision no. 657/2002/EC recommends to calculate two statistical limits, CCα and CCβ, which allow to evaluate the critical concentrations above which the method reliably distinguish and quantify a substance taking into account the variability of the method and the statistical risk to take a wrong decision. The calculation, which can be derived from the ISO standard no. 11843 is applied on a routine basis. An example displays a very simple way for evaluating the performance of an LC-MSMS method which has been validated a few years ago and is qualified onto a Micromass Quattro LCZ tandem mass spectrometer to monitor and confirm the nitrofuran metabolite residues in food from animal origin. Community Reference Laboratory for Antimicrobial Veterinary Drug Residue Control in Food from Animal Origin     

Lead‐enhanced gas‐phase stability of multiply charged EDTA anions: a combined experimental and theoretical study

Besides their fundamental importance, multiply charged anions (MCAs) are considered as promising molecular capacitors for which their intrinsic stabilities are of great significance. Herein, the gas‐phase stabilities of ethylenediaminetetraacetic acid (EDTA) anions (i.e. [EDTA‐nH]^n?, n = 1–4) and their Pb(II) complexes (i.e. [EDTA + Pb‐nH]^(2‐n)?, n = 3, 4) have been investigated using an approach that combines extractive electrospray ionization mass spectrometry (EESI‐MS) measurements, Car–Parrinello molecular dynamics simulations and density functional theory/Tao–Perdew–Staroverov–Scuseria calculations. The EESI‐MS data showed that the doubly charged EDTA anions in the form of [EDTA‐2H]^2? and [EDTA + Pb‐4H]^2? were much more abundantly observed than the singly charged species such as [EDTA‐H]^? and [EDTA + Pb‐3H]^?, respectively. The calculation results indicated that [EDTA‐2H]^2? and [EDTA + Pb‐4H]^2? anions were thermodynamically more stable than the [EDTA‐H]^? and [EDTA + Pb‐3H]^? species in the gas phase, respectively. The [EDTA + Pb‐3H]^? anions preferred five‐coordinated structure, whereas [EDTA + Pb‐4H]^2? anions formed either five‐coordinated or six‐coordinated structures. The calculations further revealed that significant electron clouds drifting from the ligand EDTA to the metal Pb(II) ions and the large distances between the carboxylic groups reduced the Coulomb repulsion among the excess electrons of these MCAs. Our data demonstrated that EESI‐MS combined with theoretic calculations were able to provide a deep insight into the fundamental behavior of stability of MCAs in the gas phase and, thus, might be useful tools for studying MCAs for potential molecular capacitors. Copyright © 2012 John Wiley & Sons, Ltd.     

Comparison of analytical methods: ICP-QMS,ICP-SFMS and FF-ET-AAS for the determination of V,Mn, Ni,Cu, As,Sr, Mo,Cd and Pb in ground natural waters

Three analytical methods, namely, inductively coupled plasma sector field mass spectrometry (ICP-SFMS); inductively coupled plasma quadrupole mass spectrometry (ICP-QMS) and filter-furnace electrothermal atomic-absorption spectroscopy (FF-ET-AAS) for the determination of V, Mn, Ni, Cu, As, Sr, Mo, Cd and Pb in ground natural water samples were compared and evaluated for their capacity to provide reliable and precise results. Two certified reference materials (SLEW-3 Estuarine Water; SLRS-4 River Water) were analysed to prove that accurate results could be obtained by using all the listed methods with properly optimised parameters. The limit of detection (LOD) for V, Mn, Ni, Cu, As, Sr, Mo, Cd and Pb provided by the ICP-MS methods ranged from 0.001 to 0.05 µg L^?1. Such LOD proved sufficient for the reliable determination of the listed elements in ground natural waters. However, the LOD of the FF-ET-AAS was approximately two orders of magnitude higher than that of ICP-MS, which made it impossible to quantify V, Mn, Ni, Mo and Pb. The effects of the usage of the collision cell mode in ICP-QMS and of the desolvation system Apex for ICP-SFMS to eliminate oxide ions levels were investigated. For all three analytical methods, the influence of the matrix effect on the results of the determination of the investigated elements using matrix model solution, external calibration and standard addition methods was evaluated. A comparison using a paired Student’s t-test between the results obtained by both ICP-MS methods for V, Mn, Ni, Cu, As, Sr, Mo, Cd and Pb concentrations in ground natural waters showed that there was no significant difference on a 95% confidence level. The precision of the results for ICP-SFMS, ICP-QMS and FF-ET-AAS varied between ~0.5 and 11; 2.5 and 12.5; 3 and 13.5%, respectively. Moreover, ICP-SFMS equipped with the desolvation system APEX proved a better choice for As, Cu and Mn analysis due to its better LOD (0.008, 0.03 and 0.02 µg L^?1, respectively) and precision (S_r ≤ 5.0; 7.5; 9.0%, respectively) compared to ICP-QMS and FF-ET-AAS.     

A semi‐quantitative approach for the rapid screening and mass profiling of naphthenic acids directly in contaminated aqueous samples

We report the use of a direct sampling, online analytical approach for the determination of acid extractable naphthenic acids in complex aqueous samples, known as condensed phase membrane introduction mass spectrometry (CP‐MIMS). The technique employs a capillary hollow fibre semi‐permeable membrane probe configured for immersion into a pH adjusted sample. A continuously flowing methanol acceptor phase transfers naphthenic acids to an electrospray ionization source, operated in negative ion mode, whereupon they are analysed by mass spectrometry as [M–H]^? ions. High‐resolution mass spectrometry is used to characterize the influence of sample pH on membrane transport of multiple components of complex naphthenic acid mixtures. We demonstrate the use of CP‐MIMS for semi‐quantitative analysis of real‐world samples using selected ion monitoring and full scan mass spectra at unit mass resolution. The technique has also been employed to continuously monitor the temporal evolution in the mass profile and concentrations of individual naphthenic acid isomer classes in heterogeneous solutions during adsorption processes. Copyright © 2015 John Wiley & Sons, Ltd.     

Identification of phase I and II metabolites of the new designer drug α‐pyrrolidinohexiophenone (α‐PHP) in human urine by liquid chromatography quadrupole time‐of‐flight mass spectrometry (LC‐QTOF‐MS)

Pyrrolidinophenones represent one emerging class of newly encountered drugs of abuse, also known as ‘new psychoactive substances’, with stimulating psychoactive effects. In this work, we report on the detection of the new designer drug α‐pyrrolidinohexiophenone (α‐PHP) and its phase I and II metabolites in a human urine sample of a drug abuser. Determination and structural elucidation of these metabolites have been achieved by liquid chromatography electrospray ionisation quadrupole time‐of‐flight mass spectrometry (LC‐ESI‐QTOF‐MS). By tentative identification, the exact and approximate structures of 19 phase I metabolites and nine phase II glucuronides were elucidated. Major metabolic pathways revealed the reduction of the ß‐keto moieties to their corresponding alcohols, didesalkylation of the pyrrolidine ring, hydroxylation and oxidation of the aliphatic side chain leading to n‐hydroxy, aldehyde and carboxylate metabolites, and oxidation of the pyrrolidine ring to its lactam followed by ring cleavage and additional hydroxylation, reduction and oxidation steps and combinations thereof. The most abundant phase II metabolites were glucuronidated ß‐keto‐reduced alcohols. Besides the great number of metabolites detected in this sample, α‐PHP is still one of the most abundant ions together with its ß‐keto‐reduced alcoholic dihydro metabolite. Monitoring of these metabolites in clinical and forensic toxicology may unambiguously prove the abuse of the new designer drug α‐PHP. Copyright © 2015 John Wiley & Sons, Ltd.     

Quantification of asenapine and three metabolites in human plasma using liquid chromatography-tandem mass spectrometry with automated solid-phase extraction: application to a phase I clinical trial with asenapine in healthy male subjects

The development and validation of methods for determining concentrations of the antipsychotic drug asenapine (ASE) and three of its metabolites [N-desmethylasenapine (DMA), asenapine-N(+) -glucuronide (ASG) and 11-O-sulfate-asenapine (OSA)] in human plasma using LC-MS/MS with automated solid-phase extraction is described. The three assessment methods in human plasma were found to be acceptable for quantification in the ranges 0.0250-20.0 ng/mL (ASE), 0.0500-20.0 ng/mL (DMA and OSA) and 0.250-50.0 ng/mL (ASG).     

Development and validation of an LC‐MS/MS method for the determination of tigecycline in human plasma and cerebrospinal fluid and its application to a pharmacokinetic study

Tigecycline (TGC) is an important antibiotic in treating various drug‐resistant bacteria. The dosage regimen for cerebral intraventricular TGC is still unknown. The aim of the study was to develop and validate liquid chromatography–tandem mass spectrometry (LC‐MS/MS) methods for the determination of TGC in human plasma and cerebrospinal fluid (CSF) to obtain an applicable regimen. The ion transitions under ESI positive model were performed at m/z 586.3 > 513.2 and m/z 595.3 > 514.3 for TGC and d9‐TGC internal standard (IS). For plasma and CSF samples, the calibration curve of TGC was linear within the ranges 25–2000 and 250–100,000 ng/mL; the IS normalized matrix effect was within the ranges 96.46–101.06% and 101.13–103.58%, respectively, for all. TGC was stable under all tested conditions. The patient received 1 mg intraventricular and 49 mg intravenous administration of TGC. The AUC_0–12 in plasma and CSF calculated according to our noncompartment model were 4713 and 23,0238 h ng/mL, respectively. Given our findings cerebral intraventricular TGC may be a choice for clinicians to treat drug‐resistant Gram‐negative bacterial‐induced meningitis and the safety and efficacy of this administration route warrants further study.     

Determination of 61 organic pollutants in drinking water by solid phase extraction followed by liquid and gas chromatography coupled to tandem mass spectrometry: an analytical strategy for a routine laboratory

A method for determination of 61 organic pollutants (polycyclic aromatic hydrocarbons and organochlorine, organophosphorous and organonitrogen pesticides) is proposed. It is based on solid phase extraction (SPE) and subsequent analysis of the extract by liquid and gas chromatography coupled to tandem mass spectrometry. Method validation yielded to the following values: limits of quantification, from 0.005 to 0.020?µg?L^?1; trueness, 95% to 113% and reproducibility (as percent relative standard deviation), 2% to 15%. Additionally, the method performed well in various proficiency tests.