Screening,library-assisted identification and validated quantification of fifteen neuroleptics and three of their metabolites in plasma by liquid chromatography/mass spectrometry with atmospheric pressure chemical ionization

A liquid chromatographic/mass spectrometric assay with atmospheric pressure chemical ionization (APCI-LC/MS) is presented for the fast and reliable screening and identification and for the precise and sensitive quantification of 15 neuroleptic (antipsychotic) drugs and three of their relevant metabolites in plasma. It allows confirmation of the diagnosis of a neuroleptic overdose and monitoring of psychiatric patients' compliance. The neuroleptics amisulpride, bromperidol, clozapine, droperidol, flupenthixol, fluphenazine, haloperidol, melperone, olanzapine, perazine, pimozide, risperidone, sulpiride, zotepine and zuclopenthixol and the pharmacologically active metabolites norclozapine, clozapine N-oxide and 9-hydroxyrisperidone were extracted from plasma using solid-phase extraction and were separated on a Merck LiChroCART column with Superspher 60 RP Select B as the stationary phase. Gradient elution was performed using aqueous ammonium formate and acetonitrile. After screening and identification in the scan mode using the authors' new LC/MS library, the neuroleptics were quantified in the selected-ion mode. The quantification assay was fully validated. It was found to be selective and proved to be linear from sub-therapeutic to over therapeutic concentrations for all analytes. The corresponding reference levels are listed. The accuracy and precision data were within the required limits. The analytes were stable in frozen plasma for at least 1 month. The method was successfully applied to several authentic plasma samples from patients treated or intoxicated with various neuroleptics. The validated LC/MS assay has proved to be appropriate for the isolation, separation, screening, identification and quantification of various neuroleptics in plasma for clinical toxicology and therapeutic drug monitoring purposes.     

Screening, library-assisted identification and validated quantification of 23 benzodiazepines, flumazenil, zaleplone, zolpidem and zopiclone in plasma by liquid chromatography/mass spectrometry with atmospheric pressure chemical ionization

A liquid chromatographic/mass spectrometric assay with atmospheric pressure chemical ionization (LC/APCI-MS) is presented for fast and reliable screening and identification and also for precise and sensitive quantification in plasma of the 23 benzodiazepines alprazolam, bromazepam, brotizolam, camazepam, chlordiazepoxide, clobazam, clonazepam, diazepam, flunitrazepam, flurazepam, desalkylflurazepam, lorazepam, lormetazepam, medazepam, metaclazepam, midazolam, nitrazepam, nordazepam, oxazepam, prazepam, temazepam and tetrazepam, triazolam, their antagonist flumazenil and the benzodiazepine BZ1 (omega 1) receptor agonists zaleplone, zolpidem and zopiclone. It allows confirmation of the diagnosis of an overdose situation and monitoring of psychiatric patients' compliance. The analytes were isolated from plasma using liquid-liquid extraction and were separated on a Merck LiChroCART column with Superspher 60 RP Select B as the stationary phase. Gradient elution was performed using aqueous ammonium formate and acetonitrile. After screening and identification in the scan mode using the authors' LC/MS library, the analytes were quantified in the selected-ion monitoring mode. The quantification assay was fully validated. It was found to be selective proved to be linear from sub-therapeutic to over therapeutic concentrations for all analytes, except bromazepam. The corresponding reference levels the assay's accuracy and precision data for all studied substances are listed. The accuracy and precision data were within the required limits with the exception of those for bromazepam. The analytes were stable in frozen plasma for at least 1 month. The validated assay was successfully applied to several authentic plasma samples from patients treated or intoxicated with various benzodiazepines or with zaleplone, zolpidem or zopiclone. It has proven to be appropriate for the isolation, separation, screening, identification and quantification of the drugs mentioned above in plasma for clinical toxicology, e.g. in cases of poisoning, and forensic toxicology, e.g. in cases of driving under the influence of drugs.     

Characterization of typical chemical background interferences in atmospheric pressure ionization liquid chromatography-mass spectrometry

The structures and origins of typical chemical background noise ions in positive atmospheric pressure ionization liquid chromatography/mass spectrometry (API LC/MS) are investigated and summarized in this study. This was done by classifying chemical background ions using precursor and product ion scans on most abundant background ions to draw a family tree of the commonly occurring chemical background ions. The possible structures and the origins of the major chemical background noise are clearly revealed in the family trees. In agreement with some suggestions in the literature, the chemical background ions studied so far can be classified mainly as either ions of contaminants (or their degradation fragments) or cluster-related ones. A significant contribution from the contaminants (airborne, from tubing and/or solvents) from plasticizer additives (phthalates, phenyl phosphates, sebacates and adipates, etc.) and silicones is concluded. These ions of contaminants can also serve as nuclei for the clustering of HPLC solvent or additives, such as water and acetic acid, thereby leading to a second family of background ions. This study explains the persistence of some chemical background noise even under fairly strong declustering conditions in API LC/MS. One of the other interesting conclusions is that there is a clear difference in structures between the chemical background ions and the protonated analytes generated under atmospheric pressure ionization. This conclusion will contribute to the on-going research efforts to exclusively remove or reduce the interference of chemical background noise in API LC/MS.     

Simultaneous identification and validated quantification of 11 oral hypoglycaemic drugs in plasma by electrospray ionisation liquid chromatography-mass spectrometry

The detection of diabetic metabolism disorders raises problems in forensic practice and sudden death with a subsequent negative autopsy finding is a common problem. In the case of an unclear hypoglycaemia, the detection of oral antidiabetics allows the differentiation of hypoglycaemia due to oral antidiabetics from that due to other reasons (insulin-induced, insulinoma). The development of an electrospray ionisation (ESI) liquid chromatography?Ctandem mass spectrometry (LC-MS/MS) procedure for the simultaneous identification and quantification of oral antidiabetics of the sulfonylurea, the glinide, the thiazolidinedione and the gliptin types in human plasma is desired. The following analytes were included: glimepiride, glibenclamide, gliquidone, glibornuride, glisoxepide, glipizide and gliclazide (sulfonylurea type), nateglinide and repaglinide (glinide type), rosiglitazone and pioglitazone (thiazolidinedione type) and the dipeptidyl peptidase inhibitors vildagliptin, sitagliptin and saxagliptin. After a liquid?Cliquid extraction with tert-butyl methyl ether at two pHs, the oral antidiabetics were separated with fast gradient elution over a C₈ column. Identification of the oral antidiabetics was achieved by two specific ion transitions of each analyte in multiple reaction monitoring mode. Quantification was performed by referring the most intense ion transition peak areas to peak areas of the ion transitions of deuterated oral antidiabetics (hydroxytolbutamide-d ₉ for the sulfonylureas, repaglinide-ethyl-d ₅ for the glinides, pioglitazone-d ₄ for the thiazolidinediones and vildagliptin-d ₃ for the gliptins). The assay was validated according to international guidelines. The LC-MS/MS assay allows the simultaneous identification of 14 oral antidiabetics and quantification of 11 oral antidiabetics in plasma in the ESI mode in a single run. Linearity is shown up to overdose concentrations. The limits of detection with a signal-noise-ratio greater than 3 were below 1?ng/ml for all analytes. Recoveries ranged from 78 to 105%; for vildagliptin and saxagliptin recoveries were worse (45%) owing to their hydrophilic character. Intraday and interday precision and accuracy were below 20% for 11 drugs at three concentrations. For the gliptins, several validation parameters were out of range and, therefore, quantitatively this method is inappropriate.     

Comparison of reversed-phase liquid chromatography-mass spectrometry with electrospray and atmospheric pressure chemical ionization for analysis of dietary tocopherols

ESI and APCI ionization techniques in both negative and positive ion modes were evaluated for simultaneous LC-MS analysis of the four tocopherol homologues (alpha, beta, gamma and delta). The ESI and APCI ionization of tocopherols in positive ion mode was not efficient and proceeded via two competitive mechanisms, with the formation of protonated pseudo-molecular ions and molecular ions, which adversely influenced the repeatability of MS signal. Ionization in negative ion mode in both ESI and APCI was more efficient as it only produced target deprotonated pseudo-molecular ions. The APCI in negative ion mode showed larger linearity range, lower detection limits and was less sensitive to the differences in chemical structure of analytes and nature of applied solvents than negative ion ESI. Negative ion APCI was, therefore, chosen for the development of LC-MS method for simultaneous determination of the four tocopherols in foods. A baseline separation of the tocopherols was achieved on novel pentafluorophenyl silica-based column Fluophase PFP. The use of methanol-water (95:5, v/v) as a mobile phase was preferable to the use of acetonitrile-water due to considerable gain in MS signal. The limits of quantifications were 9 ng/mL for alpha-tocopherol, 8 ng/mL for beta- and gamma- and 7.5 ng/mL for delta-tocopherol when 2 microL was injected. This method was successfully applied to determination of tocopherols in sunflower oil and milk.     

Effect of high-performance liquid chromatography mobile phase components on sensitivity in negative atmospheric pressure chemical ionization liquid chromatography-mass spectrometry

We have investigated the effect of several common buffers (10-mM formic acid, 10-mM ammonium acetate, and 100-mM ammonium acetate) on the ionization of a series of model compounds that are amenable to negative atmospheric pressure chemical ionization to determine the extent of ionization quenching that can occur. In addition, we have compared the sensitivity of these standard mobile phases to a mobile phase that does not contain an acidic buffer component, but rather a base (N-methylmorpholine). The results showed that, as expected, the sensitivity for the test analytes was greatest in the mobile phase that lacked acidic components. In general, ionization of analytes that contained a single, more weakly acidic functional group was inhibited to a greater degree by more strongly acidic buffer components. In some cases, ionization was quenched completely by acidic buffer components, Ionization of compounds that were more strongly acidic was quite good in all mobile phases tested. Differences in the ionization efficiencies of the analytes in each mobile phase were correlated with the gas-phase reagent ions present. As a point of reference, each of the analytes also was analyzed in the positive ion mode and the signal intensities were compared to those obtained in the negative ion mode. In addition, the utility of mobile phases that contained N-methylmorpholine for chromatographic separations was demonstrated.     

Validated assay for quantification of oxcarbazepine and its active dihydro metabolite 10-hydroxycarbazepine in plasma by atmospheric pressure chemical ionization liquid chromatography/mass spectrometry

Oxcarbazepine (OX), a new antiepileptic, may lead to unwanted side-effects or even life-threatening intoxications after overdose. Therefore, a validated liquid chromatographic/mass spectrometric (LC/MS) assay was developed for the quantification of OX and its pharmacologically active dihydro metabolite (dihydrooxcarbazepine, DOX, often named 10-hydroxycarbazepine). OX and DOX were extracted from plasma by the authors' standard liquid/liquid extraction and were separated on a Merck LiChroCART column with Superspher 60 RP Select B as the stationary phase. Gradient elution was performed using aqueous ammonium formate and acetonitrile. The compounds were quantified in the selected-ion monitoring mode using atmospheric pressure chemical ionization electrospray LC/MS. The assay was fully validated. It was found to be selective. The calibration curves were linear from 0.1 to 50 mg l(-1) for OX and DOX. Limits of quantification were 0.1 mg l(-1) for OX and DOX. The absolute recoveries were between 60 and 86%. The accuracy and precision data were within the required limits. The analytes in frozen plasma samples were stable for at least 1 month. The method was successfully applied to several authentic plasma samples from patients treated or intoxicated with OX. The measured therapeutic plasma levels ranged from 1 to 2 mg l(-1) for OX and from 10 to 40 mg l(-1) for DOX. The validated LC/MS assay proved to be appropriate for quantification of OX and DOX in plasma for clinical toxicology and therapeutic drug monitoring purposes. The assay is part of a general analysis procedure for the isolation, separation and quantification of various drugs and for their full-scan screening and identification.     

Identification of nitrate ester explosives by liquid chromatography-electrospray ionization and atmospheric pressure chemical ionization mass spectrometry

A new continuous chromatographic process (VARICOL) has been presented recently. The basic principle of the new VARICOL process consists of an asynchronous shift of the inlet/outlet lines in a multi-column system on a recycle loop. This process has been used to perform the separation of the optical isomers of the SB-553261 racemate. In this paper, we illustrate that for this specific separation, the VARICOL process is more efficient than the well-known SMB process.     

Quantification of tripdiolide in human whole blood by liquid chromatography coupled with atmospheric pressure chemical ionization tandem mass spectrometry

A liquid chromatographic-tandem mass spectrometric (LC-MS/MS) assay was developed and validated to determine tripdiolide in human whole blood using dexamethasone acetate as an internal standard (I.S.). Liquid-liquid extraction with ethyl acetate was used to isolate them from the biological matrix. Detection was performed on a mass spectrometer coupled with a negative atmospheric pressure chemical ionization (APCI) in the multiple-reaction monitoring (MRM) mode. The calibration curve was linear (r² = 0.9973) in the concentration range of 0.5-100.0 ng/mL in human whole blood with a lower limit of quantification of 0.5 ng/mL. Intra-day and inter-day relative standard deviations (R.S.D.s) were less than 7.0 and 10.1%, respectively. Extraction recoveries of tripdiolide ranged from 80.5 to 90.1%. This assay can be used to determine trace tripdiolide in human whole blood.     

Comparison of electrospray ionization,atmospheric pressure chemical ionization and atmospheric pressure photoionization for determining estrogenic chemicals in water by liquid chromatography tandem mass spectrometry with chemical derivatizations

This study compared the sensitivities and matrix effects of four ionization modes and four reversed-phase liquid chromatographic (LC) systems on analyzing estrone (E1), 17β-estradiol (E2), estriol (E3), 17α-ethinylestradiol (EE2), 4-nonylphenol (NP), 4-tert-octylphenol (OP), bisphenol A (BPA) and their derivatives of dansyl chloride or pentafluorobenzyl bromide (PFBBr) in water matrixes using a triple-quadrupole mass spectrometer with selected reaction monitoring (SRM). The four probes were electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), atmospheric pressure photoionization (APPI) and APCI/APPI; the four LC systems were ultra-performance liquid chromatography (UPLC) with or without post-column split, a mixed-mode column and two-dimensional LC (2D-LC). Dansylated compounds with ESI at UPLC condition had the most intense signals and less matrix effects of the various combinations of ionization and LC systems. The on-column limits of detection (LODs) of dansylated estrogens by SRM were 0.05–0.20 pg, and the LODs in sewage treatment plant effluent and in river water were 0.23–0.52 and 0.56–0.91 ng/L, respectively. The LODs using selected ion monitoring (SIM) reached low ng/L levels in real samples and measured concentrations were comparable with those of SRM.     

Rapid identification of Mg-chelated chlorins by on-line high performance liquid chromatography/atmospheric pressure chemical ionization mass spectrometry

On-line high performance liquid chromatography/atmospheric pressure chemical ionization mass spectrometry (HPLC/APCI-MS) has been applied to the identification of a number of tetrapyrrolic pigments as well as several magnesium-free analogs. Mass spectra, acquired both in positive (+) and negative (-) ionization mode, allow not only the determination of the structural features of the pigments, but also a very easy differentiation of the Mg-chelated pigments from the free bases. In the positive ionization mode, all pigments show mainly a [M + H]+ ion and a [M + H - C20H38]+ fragment ion corresponding to the loss of the phytyl chain via a hydrogen rearrangement. In the negative ionization mode, on the other hand, although all pigments give an abundant molecular anion [M]-*, only the Mg-chelated chlorin spectra show a prominent fragment [M - C20H39]- produced by a formal loss of the phytyl radical.     

Supercritical fluid extraction and quantification of aflatoxins in Zizyphi Fructus by liquid chromatography/atmospheric pressure chemical ionization tandem mass spectrometry

An integrated method combining supercritical fluid extraction (SFE) with liquid chromatography/atmospheric pressure chemical ionization tandem mass spectrometry (LC/APCI-MS/MS) was developed and successfully applied to quantify aflatoxins (AFs) in Zizyphi Fructus (fruits of Zizyphus jujube), a traditional Chinese medicine. To minimize the potential interferences caused by the complex matrix in Zizyphi Fructus, a SFE pretreatment was performed. In addition, electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) spectra were also compared. The results showed that the calibration curves of AFB(1), AFB(2), AFG(1), and AFG(2) were all linear over the range of concentration from 1 to 50 ng/g, the squared correlation coefficients (r(2)) were over 0.995, and the detection limits of the method were between 0.17 and 0.32 ng/g. It showed high recovery and good precision in quantitating AFs in Zizyphi Fructus without further clean-up. Further, fragmentation pathways of protonated AFs in APCI-MS/MS were clearly proposed which could predict the existence of AFB or AFG series. To test the empirical validity of the proposed methodology in this paper, eight random samples of Zizyphi Fructus collected from supermarkets and traditional Chinese medicine stores in different geographical areas of Taiwan were analyzed. The results indicated that low levels of AFs were detected in only one of them.     

Analysis of pyridoquinoline derivatives by liquid chromatography/atmospheric pressure chemical ionization mass spectrometry

A method using liquid chromatography/atmospheric pressure chemical ionization mass spectrometry (LC/APCI-MS) has been developed for the characterization and determination of pyridoquinoline derivatives 4,6-bis(dimethylaminoethylamino)-2,8,10-trimethylpyrido[3,2-g]quinoline, 4,6-bis(dimethylaminoethoxy)-2,8,10-trimethylpyrido[3,2-g]quinoline and 4,6-bis[(dimethylaminoethyl)thio]-2,8,10-trimethylpyrido[3,2-g] quinoline, all with potential antitumor properties. LC separation was performed on a conventional C18 column using a binary mobile phase composed of acetonitrile and 50 mM aqueous ammonium formate at pH 3. The APCI mass spectra obtained showed that proton addition giving [M + H]+ was the common mode of ionization to the amino- and thiopyridoquinolines, whereas the alkoxypyridoquinoline was identified by the main formation of the [M - (C2H3)N(CH3)2 + H]+, followed by the [M + H]+ ion. The LC separation conditions and MS detection parameters were optimized for the determination. The analytical method was also applied to the determination of these pyridoquinoline derivatives in fetal calf serum using liquid-liquid extraction with dichloromethane. Acceptable recovery values were obtained, ranging between 45 and 98%.     

Stereochemistry of norditerpenoid alkaloids by liquid chromatography/atmospheric pressure chemical ionization mass spectrometry

High-performance liquid chromatography/atmospheric pressure chemical ionization mass spectrometry (HPLC/APCI-MS) is a very promising approach to structural investigations of positional isomers and stereoisomers. This method was applied successfully to stereoisomeric norditerpenoid alkaloids differing in configuration at C-6. APCI-MS allowed the easy and precise control of energy deposition by varying the drift voltage. Comparison of the breakdown curves, observed by changing the potential difference between the first electrode and the second electrode of the APCI ion source, revealed the stereochemical dependence of different fragmentations. Comparison of the APCI spectra showed that the abundance of fragment ions was significantly higher for C-6beta alkaloid than for C-6alpha alkaloid. The axial positions of the corresponding substituents (6-methoxyl and 8-hydroxyl) strongly suggested a 1,3-diaxial interaction effect of the fragmentation. The characteristic fragment ions were formed by the loss of water or acetic acid at position 8, irrespective of the stereochemistry at position 6. The possibility of distinct fragmentation mechanisms depending on the stereochemistry of the precursor ion could be discerned by recording the spectra in a deuterated solvent system of 0.05 M ammonium acetate in D2O-acetonitrile-tetrahydrofuran. Loss of D2O from the precursor ion gave the fragment ion. This result indicated that the proton of protonation was included in the leaving water molecule. The peak intensity ratio R = [M+H]+/[M+H-H2O]+ manifested the stereochemical differentiation of alkaloids at position 6.     

Optimization of the atmospheric pressure chemical ionization liquid chromatography mass spectrometry interface

Use of optimized instrument parameters that result from statistical experimentation revealed that the sensitivity of atmospheric pressure chemical ionization (APCI) liquid chromatography-mass spectrometry (LC/MS) is greater than the sensitivity of an optimized Thermabeam? LC/MS interface by about 3 orders of magnitude, when tested on aromatic compounds. APCI is one of the few LC/MS techniques in which the chromatogram is directly comparable with liquid chromatographs that use ultraviolet detection. The optimum instrument parameters for a Finnigan SSQ-7000 APCI LC/MS interface were found at low flow rates (e. g., 0. 1 mL/min), relatively low capillary heat (e. g., 225 °C), and high sheath-gas pressure (e. g., 60 lb/in²). The optimization was achieved by monitoring the responses of sensitivity, fragmentation, and cluster ion formation. The fine tuning for high sensitivity calls for a high percentage of water in the mobile phase. In contrast, a high percentage of organic content in the mobile phase is required to obtain abundant protonated molecular ions with respect to fragmentation and clustering. This is an important consideration for analyses of unknowns.     

Screening of steroids in horse urine and plasma by using electron impact and chemical ionization gas chromatography-mass spectrometry

Gas chromatography with chemical ionization mass spectrometry and selected-ion monitoring provided a sensitive method for the screening and confirmation of steroids in horse urine and plasma. Chemical ionization mass spectrometry was more sensitive than the electron impact ionization mass spectrometry for most of the steroids except for testosterone, prednisone-metabolite-2 and prednisolone-metabolite-2. The chromatographic conditions used in this study provided clean separation of different natural and synthetic steroids. Approximately 75-85% of the steroids added to plasma and approximately 65-70% of the steroids added to urine were recovered by the extraction procedure used in this study.     

Comprehensive chemical profiling and quantification of Shexiang Xintongning tablets by integrating liquid chromatography-mass spectrometry and gas chromatography-mass spectrometry

Shexiang Xintongning tablet (SXXTN) is a traditional Chinese medicine (TCM) preparation for the treatment of coronary heart disease (CHD) angina pectoris. However, due to the complexity of the compounds in SXXTN, the active chemical components responsible for the therapeutic effect are still ambiguous. The purpose of our study was to characterize the chemical profile of SXXTN and quantify the representative chemicals. The high-performance liquid chromatography coupled with time-of-flight mass spectrometry (HPLC-QTOF MS) method and gas chromatograph coupled with mass spectrometry (GC–MS) method were utilized to identify the chemical constituents of SXXTN. A total of 140 compounds including alkaloids, ginsenosides, organic acids, esters, triterpenes, phthalides and amino acid were identified in accordance with their retention times, accurate masses and characteristic MS/MS fragment patterns. Forty-four volatile components were characterized by GC–MS through NIST database matching. In the further research of quantitative analysis, 40 non-volatile compounds and 17 volatile compounds were determined and successfully applied for detecting in 7 batches of SXXTN samples by high performance liquid chromatography coupled with triple-quadrupole tandem mass spectrometry (HPLC-QQQ MS) and gas chromatograph coupled with triple-quadrupole tandem mass spectrometry (GC-QQQ MS) in multiple reaction monitoring (MRM) mode, respectively. The quantitative methods were verified in linearity, precision, repeatability stability and recovery. The above results indicated that the established method was practical and reliable for synthetical quality evaluation of SXXTN. In addition, our study might supplement the chemical evidence for disclosing the material basis of its therapeutic effects.     

Determination of chlorantraniliprole residues in crops by liquid chromatography coupled with atmospheric pressure chemical ionization mass spectrometry/mass spectrometry

An analytical method is presented for the determination of chlorantraniliprole residues in crops. Chlorantraniliprole residues were extracted from crop matrixes with acetonitrile after a water soak. The extracts were passed through a strong anion-exchange (SAX) SPE cartridge stacked on top of a reversed-phase (RP) polymer cartridge. After both cartridges were rinsed and vacuum-dried, the SAX cartridge was removed, and chlorantraniliprole was eluted from the RP polymer cartridge with acetonitrile. The acetonitrile eluate was evaporated to dryness, reconstituted, and analyzed using an LC/MS/MS instrument equipped with an atmospheric pressure chemical ionization source. The method was successfully validated at 0.010, 0.10, and 10 mg/kg for the following crop matrixes: potatoes, sugar beets (tops), lettuce, broccoli, soybeans, soybean forage, tomatoes, cucumbers, oranges, apples, pears, peaches, almonds (nutmeat), rice grain, wheat grain, wheat hay, corn stover, alfalfa forage, cottonseed, grapes, and corn grain. The average recoveries from all crop samples fortified at the method LOQ ranged from 91 to 108%, with an overall average recovery of 97%. The average recoveries from all crop samples fortified at 10 times the method LOQ ranged from 89 to 115%, with an overall average recovery of 101%. For all of the fortified control samples analyzed in this study, the overall average recovery was 99%.