Mono- and diiodocyclophosphamide as possible internal standards for cyclophosphamide quantification: characterization by ion trap multi-stage mass spectrometry and effects of iodine-chlorine substitution on the fragmentation pattern

Hospital personnel involved in antineoplastic drug preparation and administration to patients are exposed to large amounts of these drugs. Labour legislation indicates the necessity of planning monitoring strategies aimed at prevention and/or reduction of drug exposure. Monitoring strategies consist of quantitative determinations of indicators, present in environmental and biological matrices. Among the antineoplastic drugs widely used, cyclophosphamide (CP) has been identified as a suitable indicator of potential exposure to mixtures of antineoplastic drugs. Many literature methods for quantitative analysis of CP involve either liquid (LC) or gas chromatography (GC) with mass spectrometry (MS), both of which require use of a suitable internal standard. The present work focuses on the synthesis of mono- and diiodocyclophosphamide (CPI and CPI(2)) to be used as internal standard. These compounds were analyzed by GC/EI-MS/MS and LC/ESI-MS(n) using ion trap mass spectrometry. The product ion mass spectra are interpreted in terms of proposed structures of fragment ions. Iodine-chlorine substitution resulted in a weakening of the carbon-halogen bond with a noteworthy influence on the ion fragmentation processes. The proposed suitability of CPI and CPI(2) as internal standards was based on similarities to CP as regards ionization and fragmentation processes. The results obtained suggest that CPI could be used as internal standard for CP quantification by LC/ESI-MS/MS, and CPI(2) for GC/EI-MS/MS analyses.     

Applications of silver nanoparticles capped with different functional groups as the matrix and affinity probes in surface-assisted laser desorption/ionization time-of-flight and atmospheric pressure matrix-assisted laser desorption/ionization ion trap mass spectrometry for rapid analysis of sulfur drugs and biothiols in human urine

A strategy is presented for the analysis of sulfur drugs and biothiols using silver nanoparticles (AgNPs) capped with different functional groups as the matrix and affinity probes in surface-assisted laser desorption/ionization time-of-flight mass spectrometry (SALDI-TOF MS) and atmospheric pressure-matrix assisted laser desorption/ionization ion trap mass spectrometry (AP-MALDI-ITMS). Biothiols adsorbed on the surface of AgNPs through covalent bonding were subjected to ultraviolet (UV) radiation that enabled desorption and ionization due to the excellent photochemical property of NPs. The proposed method has been successfully applied for the determination of cysteine and homocysteine in human urine samples using an internal standard. The limit of detection (LOD) and limit of quantification (LOQ) for cysteine and homocysteine in urine sample are 7 and 22 nM, respectively, with a relative standard deviation (RSD) of <10%. The advantages of the present method compared with the methods reported in the literature for biothiol analysis are simplicity, rapidity and sensitivity without the need for time-consuming separation and tedious preconcentration processes. Additionally, we also found that the bare AgNPs can be directly used as the matrix in MALDI-TOF MS for the analysis of sulfur drugs without the addition of an extra proton source. Copyright (c) 2008 John Wiley & Sons, Ltd.     

Evaluation of four mass spectrometric methods for the gas chromatographic analysis of polychlorinated n-alkanes

The suitability of four mass spectrometric methods for the gas chromatographic analysis of polychlorinated n-alkanes (PCAs, also called chlorinated paraffins) was evaluated and compared using spiked and fish liver samples. Electron ionization tandem mass spectrometry (EI-MS/MS) as well as electron capture negative ionization (ECNI) combined with low and high resolution mass spectrometry and CH4/CH2Cl2-negative ion chemical ionization (NICI) low resolution mass spectrometry were investigated. All methods showed an accuracy of <21% for the analysis of spiked fish samples. However, the analysis of real samples showed deviations of up to 46% between the four mass spectrometric methods. The influence of the selected reference standard on quantification was also evaluated. The use of a quantification standard with a degree of chlorination deviating from that of the sample can result in differences of > 100% for the ECNI methods. EI-MS/MS and CH4/CH2Cl2-NICI led to errors of maximum 17% and 33%, respectively, independent from the degree of chlorination of the used reference standard.     

Electrospray ionization fourier transform mass spectrometry quantification of enkephalin using an internal standard.

Fourier transform mass spectrometry (FTMS), long-known for its capabilities in structural characterization of molecules, is an emerging tool in quantification, and quantification methods using external and internal standards with electrospray ionization (ESI) FTMS have recently been demonstrated. Here, commercial ESI-FTMS is used to quantify the opioid pentapeptide methionine enkephalin using an internal standard. Linear working curves over three orders of magnitude are obtained using the internal standard, an improvement of one order of magnitude over the previous external standard ESI-FTMS quantification method for enkephalins. Low coefficients of variation (generally <6%) are observed, and inter-day and intra-day assays are compared and found to possess similar linearity and precision. The high mass accuracy advantage of FTMS can be exploited to give molecular specificity. Efforts to improve mass accuracy using internal mass calibration generally provide mass accuracies within 2.5 ppm.     

Rapid MALDI-MS Assays for Drug Quantification in Biological Matrices: Lessons Learned,New Developments,and Future Perspectives

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has rarely been used in the field of therapeutic drug monitoring, partly because of the complexity of the ionization processes between the compounds to be quantified and the many MALDI matrices available. The development of a viable MALDI-MS method that meets regulatory guidelines for bioanalytical method validation requires prior knowledge of the suitability of (i) the MALDI matrix with the analyte class and properties for ionization, (ii) the crystallization properties of the MALDI matrix with automation features, and (iii) the MS instrumentation used to achieve sensitive and specific measurements in order to determine low pharmacological drug concentrations in biological matrices. In the present hybrid article/white paper, we review the developments required for the establishment of MALDI-MS assays for the quantification of drugs in tissues and plasma, illustrated with concrete results for the different steps. We summarize the necessary parameters that need to be controlled for the successful development of fully validated MALDI-MS methods according to regulatory authorities, as well as currently unsolved problems and promising ways to address them. Finally, we propose an expert opinion on future perspectives and needs in order to establish MALDI-MS as a universal method for therapeutic drug monitoring.     

A liquid chromatography/tandem mass spectrometry assay for the analysis of atomoxetine in human plasma and in vitro cellular samples

A simple, rapid and sensitive method for quantification of atomoxetine by liquid chromatography–tandem mass spectrometry (LC‐MS/MS) was developed. This assay represents the first LC‐MS/MS quantification method for atomoxetine utilizing electrospray ionization. Deuterated atomoxetine (d3‐atomoxetine) was adopted as the internal standard. Direct protein precipitation was utilized for sample preparation. This method was validated for both human plasma and in vitro cellular samples. The lower limit of quantification was 3 ng/mL and 10 nm for human plasma and cellular samples, respectively. The calibration curves were linear within the ranges of 3–900 ng/mL and 10 nm to 10 µm for human plasma and cellular samples, respectively (r² > 0.999). The intra‐ and inter‐day assay accuracy and precision were evaluated using quality control samples at three different concentrations in both human plasma and cellular lysate. Sample run stability, assay selectivity, matrix effect and recovery were also successfully demonstrated. The present assay is superior to previously published LC‐MS and LC‐MS/MS methods in terms of sensitivity or the simplicity of sample preparation. This assay is applicable to the analysis of atomoxetine in both human plasma and in vitro cellular samples. Copyright © 2012 John Wiley & Sons, Ltd.     

Development and validation of stable‐isotope dilution liquid chromatography–tandem mass spectrometric method for determination of salivary progesterone

A method for the quantification of progesterone (PROG) in human saliva using liquid chromatography–electrospray ionization–tandem mass spectrometry (LC‐ESI‐MS/MS) has been developed and validated. The saliva was deproteinized with acetonitrile, purified using a Strata™‐X cartridge, and subjected to LC‐ESI‐MS/MS. Quantification was based on selected reaction monitoring, and deuterated PROG was used as the internal standard. This method allowed the reproducible (intra‐ and inter‐assay relative standard deviations, <2.2%) and accurate (analytical recovery, 96.6–99.7%) quantification of the salivary PROG using a 400 μL sample, and the limit of quantification was 12.5 pg/mL. The developed method enabled detection of the variation in the salivary PROG concentrations of healthy volunteers during the menstrual cycle and measurement of the salivary concentrations of pregnant women. The method is expected to be an alternative to the blood PROG monitoring in clinical examinations, because saliva collection is easy, non‐invasive and repeatable. Copyright © 2011 John Wiley & Sons, Ltd.     

Quantitative determination of acetylcholine in microdialysis samples using liquid chromatography/atmospheric pressure spray ionization mass spectrometry

A fast, simple and sensitive liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed for the determination of acetylcholine in rat brain microdialysis samples. The chromatographic separation was achieved in 3 min on a reversed-phase column with isocratic conditions using a mobile phase containing 2% (v/v) of acetonitrile and 0.05% (v/v) of trifluoroacetic acid (TFA). A stable isotope-labeled internal standard was included in the analysis and detection was carried out with a linear ion trap mass spectrometer using selected reaction monitoring (SRM). Analyte ionization was performed with an atmospheric pressure chemical ionization (APCI) source without applying discharge current (atmospheric pressure spray ionization). This special ionization technique offered significant advantages over electrospray ionization for the analysis of acetylcholine with reversed-phase ion-pairing chromatography. The lower limit of quantification was 0.15 nM (1.5 fmol on-column) and linearity was maintained over the range of 0.15-73 nM, providing a concentration range that is significantly wider than that of the existing LC/MS methods. Good accuracy and precision were obtained for concentrations within the standard curve range. The method was validated and has been used extensively for the determination of acetylcholine in rat brain microdialysis samples.     

Determination of acetaldehyde in tobacco smoke using N-methyl-4-hydrazino-7-nitrobenzofurazan and liquid chromatography/mass spectrometry

A liquid chromatography/mass spectrometry (LC/MS) method for the determination of carbonyl compounds based on derivatization with N-methyl-4-hydrazino-7-nitrobenzofurazan (MNBDH) has been developed. Atmospheric pressure chemical ionization (APCI) in the positive mode proved the most versatile ionization technique for MNBD-hydrazones. APCI/MS spectra were recorded and the detection limits were determined for [M+H]⁺. ¹³C₂ acetaldehyde MNBD-hydrazone has been synthesized and characterized. It is applied as internal standard for the quantification of acetaldehyde. Tobacco smoke has been investigated concerning its carbonyl content. Acetaldehyde was identified as main product and quantified by LC/MS using internal standardization. The result is in good agreement to quantification data obtained with UV/vis detection.     

A new high-performance liquid chromatographic/electrospray ionization tandem mass spectrometric method for the simultaneous determination of cyclophosphamide, methotrexate and 5-fluorouracil as markers of surface contamination for occupational exposure monitoring

A new high-performance liquid chromatographic/electrospray ionization tandem mass spectrometric (HPLC/ESI-MS/MS) method was developed for the simultaneous quantification of 5-fluorouracil (5FU), methotrexate (MTX) and cyclophosphamide (CP) in environmental samples. These compounds, commonly used in the treatment of cancer, are recognized as genotoxic. In order to estimate the occupational exposure of hospital personnel handling these drugs, wipe samples were taken from the working surfaces and directly analyzed (with trophosphamide as internal standard) using a reversed-phase capillary column and MS/MS detection. This is the first HPLC/MS/MS method for the simultaneous determination of 5FU, MTX and CP. The present method offers high sensitivity, with detection limits of 1.1 microg l(-1) for MTX and CP and 33.3 microg l(-1) for 5FU, avoiding any sample preconcentration procedure. Rapidity, specificity, high accuracy (mean values between 92.4 and 99.9%) and precision (mean RSD values between 3.4 and 12.1%) make the method suitable for the routine determination of these three antineoplastic drugs.     

Determination of acetaldehyde in tobacco smoke using N-methyl-4-hydrazino-7-nitrobenzofurazan and liquid chromatography/mass spectrometry

A liquid chromatography/mass spectrometry (LC/MS) method for the determination of carbonyl compounds based on derivatization with N-methyl-4-hydrazino-7-nitrobenzofurazan (MNBDH) has been developed. Atmospheric pressure chemical ionization (APCI) in the positive mode proved the most versatile ionization technique for MNBD-hydrazones. APCI/MS spectra were recorded and the detection limits were determined for [M+H]+, 13C2 acetaldehyde MNBD-hydrazone has been synthesized and characterized. It is applied as internal standard for the quantification of acetaldehyde. Tobacco smoke has been investigated concerning its carbonyl content. Acetaldehyde was identified as main product and quantified by LC/MS using internal standardization. The result is in good agreement to quantification data obtained with UV/vis detection.     

Determination of Atenolol in Pharmaceutical Preparations by Gas Chromatography with Flame Ionization and Mass Spectrometric Detection

The present work describes the methodology and validation of gas chromatography with flame ionization (FID) and mass spectrometric (MS) detection after derivatization with N-Methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) for determination of atenolol with an internal standard (metoprolol) in pharmaceutical preparations. The linearity was established over the concentration range of 0.5–20 μg/mL for GC/FID and 12.5–500 ng/mL for GC/MS method. The intra- and inter-day relative standard deviation was less than 4.72 and 5.80%, respectively. Limit of quantification was determined as 500 ng/mL and 12.5 ng/mL for GC/FID and GC/MS, respectively. No interference was found from tablet excipients at the selected assay conditions. Developed GC/FID and GC/MS methods in this study are accurate, sensitive, and precise and can be easily applied to Tensinor tablet as pharmaceutical preparation.     

Switching from an analogous to a stable isotopically labeled internal standard for the LC-MS/MS quantitation of the novel anticancer drug Kahalalide F significantly improves assay performance

The importance of a stable isotopically labeled (SIL) internal standard for the quantitative LC-MS/MS assay for Kahalalide F in human plasma is highlighted. Similar results can be expected for other LC-MS/MS assays. Therefore, we emphasize the need for an SIL internal standard for accurate and precise LC-MS/MS assays of drugs in biological matrices.     

An analytical investigation of 24 oxygenated-PAHs (OPAHs) using liquid and gas chromatography–mass spectrometry

We developed two independent approaches for separation and quantitation of 24 oxygenated polycyclic aromatic hydrocarbons (OPAHs) using both liquid chromatography-atmospheric pressure chemical ionization/mass spectrometry (LC-APCI/MS) and gas chromatography-electron impact/mass spectrometry (GC-EI/MS). Building on previous OPAH research, we examined laboratory stability of OPAHs, improved existing method parameters, and compared quantification strategies using standard addition and an internal standard on an environmental sample. Of 24 OPAHs targeted in this research, 19 compounds are shared between methods, with 3 uniquely quantitated by GC-EI/MS and 2 by LC-APCI/MS. Using calibration standards, all GC-EI/MS OPAHs were within 15 % of the true value and had less than 15 % relative standard deviations (RSDs) for interday variability. Similarly, all LC-APCI/MS OPAHs were within 20 % of the true value and had less than 15 % RSDs for interday variability. Instrument limits of detection ranged from 0.18 to 36 ng mL^?1 on the GC-EI/MS and 2.6 to 26 ng mL^?1 on the LC-APCI/MS. Four standard reference materials were analyzed with each method, and we report some compounds not previously published in these materials, such as perinaphthenone and xanthone. Finally, an environmental passive sampling extract from Portland Harbor Superfund, OR was analyzed by each method using both internal standard and standard addition to compensate for potential matrix effects. Internal standard quantitation resulted in increased precision with similar accuracy to standard addition for most OPAHs using 2-fluoro-fluorenone-¹³C as an internal standard. Overall, this work improves upon OPAH analytical methods and provides some considerations and strategies for OPAHs as focus continues to expand on this emerging chemical class.

A high-performance liquid chromatography/tandem mass spectrometry method for the determination of artemisinin in rat plasma

Artemisinin is a widely used antimalarial drug. To evaluate the pharmacokinetics of artemisinin in rats, a sensitive and specific liquid chromatography/tandem mass spectrometric (LC/MS/MS) method was developed and validated for the determination of artemisinin in rat plasma. For detection, a Sciex API 4000 LC/MS/MS instrument with an electrospray ionization (ESI) TurboIonSpray inlet in the positive ion multiple reaction monitoring (MRM) mode was used to monitor precursor ([M+NH4]+) --> product ions of m/z 300.4 --> 209.4 for artemisinin and m/z 316.4 --> 163.4 for artemether, the internal standard (IS). The plasma samples were pretreated by a simple liquid-liquid extraction with ether. The standard curve was linear (r > 0.99) over the artemisinin concentration range of 1.0-200.0 ng/mL in plasma. The method had a lower limit of quantification of 1.0 ng/mL for artemisinin in 100 microL of plasma, which offered a satisfactory sensitivity for the determination of artemisinin. The intra- and inter-day precisions were measured to be within +/-5.3% and accuracy between -2.6% and 1.2% for all quality control samples, lower limit of quantification and upper limit of quantification samples. The extraction recoveries of artemisinin and the IS were 95.4 +/- 4.5% and 92.8 +/- 3.9%, respectively. This present method was successfully applied to the characterization of the pharmacokinetic profile of artemisinin in rats after oral administration.     

Importance of using highly pure internal standards for successful liquid chromatography/tandem mass spectrometric bioanalytical assays

Internal standards (IS) with similar physicochemical properties to the analyte provide multiple advantages in liquid chromatography/tandem mass spectrometric (LC/MS/MS) bioanalytical methods such as: reduction of the analysis run time, improvement in the intra‐injection reproducibility, impact reduction of matrix and ionization effects. However, it is important to evaluate the purity of the IS prior to their use. Indeed, a minor impurity in the IS may lead to an important issue during bioanalytical method development. Stable labelled internal standards are usually appropriate IS for bioanalysis. The use of oxycodone‐D3, ursodiol‐D5 and atovaquone‐D4 as internal standards in three different bioanalytical methods was evaluated. During oxycodone, oxymorphone and noroxycodone simultaneous quantification method development, oxymorphone was identified as a contaminant in oxycodone‐D3. Since the limit of quantification for oxymorphone was very low (10 pg/mL), the presence of an even low percentage of oxymorphone in oxycodone‐D3 leads to the change of the stable labelled IS for an analogue, ethylmorphine. 23‐Nordeoxycholic acid was preferred to ursodiol‐D5 as internal standard for the ursodiol, tauroursodiol and glycoursodiol simultaneous quantification method. Indeed, more than 7% of ursodiol was identified in the ursodiol‐D5 which could not be bypassed by decreasing the IS concentration without compromising the linearity. An atovaquone‐D4 reference standard revealed the non‐negligible presence of atovaquone‐D5 to atovaquone‐D8 that has a large impact on the method validation. Therefore, atovaquone‐D4 was sent for recertification since its isotopic purity was found to be much less than the isotopic purity mentioned on its certificate of analysis. Consequently, during bioanalytical method development, the purity of the IS should be scrutinized. Copyright © 2009 John Wiley & Sons, Ltd.     

Quantification of trandolapril and its metabolite trandolaprilat in human plasma by liquid chromatography/tandem mass spectrometry using solid-phase extraction

A sensitive high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (MS/MS) method was developed and validated for the simultaneous quantification of trandolapril and its metabolite trandolaprilat in human plasma using ramipril as an internal standard. Following solid-phase extraction, the analytes were separated using an isocratic mobile phase on a reversed-phase column and analyzed by MS/MS in the multiple reaction monitoring mode using the respective [M-H]- ions, m/z 429/168 for trandolapril, m/z 401/168 for trandolaprilat and m/z 415/166 for the internal standard. The method exhibited a linear dynamic range of 20-10,000 pg/mL for both trandolapril and trandolaprilat in human plasma. The lower limit of quantification was 20 pg/mL for both trandolapril and its metabolite. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. A run time of 2.0 min for each sample made it possible to analyze more than 400 human plasma samples per day. The validated method has been successfully used to analyze human plasma samples for application in pharmacokinetic, bioavailability or bioequivalence studies.     

Rapid and simple liquid chromatography tandem mass spectrometry method for the quantification of zidovudine in rat plasma

A high-performance liquid chromatography/electrospray ionization tandem mass spectrometry method was developed and validated for the quantification of zidovudine in rat plasma. Following solid-phase extraction, the analytes were separated using an isocratic mobile phase on a reverse phase column and analyzed by MS/MS in the multiple reaction monitoring mode using the respective [M+H]+ ions, m/z 268/127 for zidovudine and m/z 230/112 for the internal standard. The method exhibited a linear dynamic range of 5-500 ng/mL for zidovudine in rat plasma. The lower limit of quantification was 5 ng/mL with a relative standard deviation of less than 8%. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. A run time of 1.5 min for each sample made it possible to analyze more than 400 plasma samples per day. The validated method was applied for pharmacokinetic studies of the novel drug delivery systems of zidovudine in rats.     

Determination of famotidine in low-volume human plasma by normal-phase liquid chromatography/tandem mass spectrometry

A rapid, sensitive and robust assay procedure using liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) for the determination of famotidine in human plasma and urine is described. Famotidine and the internal standard were isolated from plasma samples by cation-exchange solid-phase extraction with benzenesulfonic acid (SCX) cartridges. The urine assay used direct injection of a diluted urine sample. The chromatographic separation was accomplished by using a BDS Hypersil silica column with a mobile phase of acetonitrile-water containing trifluoroacetic acid. The MS/MS detection of the analytes was set in the positive ionization mode using electrospray ionization for sample introduction. The analyte and internal standard precursor-product ion combinations were monitored in the multiple-reaction monitoring mode. Assay calibration curves were linear in the concentration range 0.5--500 ng ml(-1) and 0.05--50 microg ml(-1) in plasma and urine, respectively. For the plasma assay, a 100 microl sample aliquot was subjected to extraction. To perform the urine assay, a 50 microl sample aliquot was used. The intra-day relative standard deviations at all concentration levels were <10%. The inter-day consistency was assessed by running quality control samples during each daily run. The limit of quantification was 0.5 ng ml(-1) in plasma and 0.05 microg ml(-1) in urine. The methods were utilized to support clinical pharmacokinetic studies in infants aged 0-12 months.     

Ion trap liquid chromatography/tandem mass spectrometry analysis of leukotriene B4 in exhaled breath condensate

The objective of this study is the measurement of leukotriene B7 (LTB4), a potent inflammatory mediator, in exhaled breath condensate by using liquid chromatography/mass spectrometry (LC/MS and LC/MS/MS). Condensation of exhaled breath is a non-invasive method to collect airway secretions. Deuterated (d4)-LTB4 was used as internal standard. The MS and MS/MS behavior of LTB4 and LTB4-d4 was studied by electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) in both positive and negative ion polarity mode. Preliminary results show that monitoring negative ions in ESI mode has the best sensitivity for both LTB4 and LTB4-d4. Therefore, negative ESI was chosen, and the [M-H]- ions at m/z 335 and 339 were selected for quantification. The lower limit of quantification for LTB4, expressed as the lowest point of the calibration curve, was 100 pg/mL. Using this technique, we measured LTB4 in exhaled breath condensate in two healthy subjects, four asthmatic patients on anti-inflammatory treatment, and four asthmatic patients who were not on anti-inflammatory drugs. Exhaled LTB4 concentrations were detected only in asthmatic patients who were not on anti-inflammatory therapy. This method is potentially useful for non-invasive assessment of airway inflammation, but the sensitivity of the technique needs to be improved.