Profiling degradants of paclitaxel using liquid chromatography–mass spectrometry and liquid chromatography–tandem mass spectrometry substructural techniques

A rapid and systematic strategy based on liquid chromatography–mass spectrometry (LC–MS) profiling and liquid chromatography–tandem mass spectrometry (LC–MS–MS) substructural techniques was utilized to elucidate the degradation products of paclitaxel, the active ingredient in Taxol. This strategy integrates, in a single instrumental approach, analytical HPLC, UV detection, full-scan electrospray MS, and MS–MS to rapidly and accurately elucidate structures of impurities and degradants. In these studies, degradants induced by acid, base, peroxide, and light were profiled using LC–MS and LC–MS–MS methodologies resulting in an LC–MS degradant database which includes information on molecular structures, chromatographic behavior, molecular mass, and MS–MS substructural information. The stressing conditions which may cause drug degradation are utilized to validate the analytical monitoring methods and serve as predictive tools for future formulation and packaging studies. Degradation products formed upon exposure to basic conditions included baccatin III, paclitaxel sidechain methyl ester, 10-deacetylpaclitaxel, and 7-epipaclitaxel. Degradation products formed upon exposure to acidic conditions included 10-deacetylpaclitaxel and the oxetane ring opened product. Treatment with hydrogen peroxide produced only 10-deacetylpaclitaxel. Exposure to high intensity light produced a number of degradants. The most abundant photodegradant of paclitaxel corresponded to an isomer which contains a C3–C11 bridge. These methodologies are applicable at any stage of the drug product cycle from discovery through development. This library of paclitaxel degradants provides a foundation for future development work regarding product monitoring, as well as use as a diagnostic tool for new degradation products.     

Quantification of lysophosphatidic acids in rat brain tissue by liquid chromatography–electrospray tandem mass spectrometry

Lysophosphatidic acid (LPA) is a lipid mediator with multiple biological functions. A highly selective and sensitive liquid chromatography–tandem mass spectrometry (LC/MS/MS) method was developed for the determination of LPAs (16:0 LPA, 18:0 LPA, 18:1 LPA, 20:4 LPA) in rat brain cryosections. After partitioning the LPAs from other lipophilic material present in the tissue with a liquid–liquid extraction, a reversed-phase column and ion pair technique was used for separating analytes with a gradient elution. An internal standard (17:0 LPA) was included in the analysis. Detection and quantification of the LPAs were carried out with a triple quadrupole mass spectrometer using negative electrospray ionization (ESI) and multiple reaction monitoring (MRM). The artificial formation of LPAs from lysophosphatidylcholines during the sample preparation procedure and instrumentation was carefully studied during the method development. The method was validated; acceptable selectivity, accuracy, precision, recovery, and stability were obtained for concentrations within the calibration curve range of 0.02–1.0 μM for LPAs. The quantification limit of the assay was 54 fmol injected into column for each LPAs. The method was applied to comparative studies of LPA levels in rat brain cryosections after the various chemical pre-treatments of the sections.     

Stable isotope labeling by amino acids in cell culture-based liquid chromatography–mass spectrometry assay to measure microtubule dynamics in neuronal cell cultures

Microtubules (MTs) are highly dynamic polymers composed of α- and β-tubulin heterodimers. Dysregulation of MT dynamics in neurons may be a contributing factor in the progression of various neurodegenerative diseases. We developed a stable isotope labeling by amino acids in cell culture (SILAC)-based liquid chromatography–mass spectrometry (LC–MS) method to measure the fraction of [¹³C₆]leucine-labeled α-tubulin-derived surrogate peptides. Using this approach, we measured the time course of incorporation of [¹³C₆]leucine label into the MT and dimer pools isolated from cycling cells and rat primary hippocampal neurons. We found that the MT pool is in rapid equilibrium with the dimer pool in the cycling cells, consistent with rapid MT polymerization/depolymerization during cell proliferation. Conversely, in neurons, we found that labeling of the MT pool was rapid, whereas the dimer pool was delayed. These results suggest that newly synthesized α-tubulin is first incorporated into MTs or complexes that co-sediment with MTs and that appearance of labeled α-tubulin in the dimer pool may be a consequence of MT depolymerization or breakdown. Our results demonstrate that a SILAC-based approach can be used to measure MT dynamics and may have utility for exploring MT dysregulation in various models of neurodegenerative disease.     

Analysis of corticosteroids in equine urine by liquid chromatography–mass spectrometry

A liquid chromatography–mass spectrometry (LC–MS) method for the analysis of corticosteroids in equine urine was developed. Corticosteroid conjugates were hydrolysed with β-glucuronidase; free and enzyme-released corticosteroids were then extracted from the samples with ethyl acetate followed by a base wash. The isolated corticosteroids were detected by LC–MS and confirmed by LC–MS–MS in the positive atmospheric pressure chemical ionisation mode. Twenty-three corticosteroids (comprising hydrocortisone, deoxycorticosterone and 21 synthetic corticosteroids), each at 5 ng/ml in urine, could easily be analysed in 10 min.     

Emerging liquid chromatography–mass spectrometry technologies improving dried blood spot analysis

Dried blood spots (DBS), a micro blood sampling technique, has recently gained interest in drug discovery and development due to its inherent advantages over the conventional whole blood, plasma or serum sample collection. Since the regulatory authorities have agreed to the use of blood as an acceptable biological matrix for drug exposure measurements, its applications have been extended not only to therapeutic drug monitoring but also to toxicokinetic and pharmacokinetic studies. The pharmaceutical industry is keen to promote DBS as a prominent tool in bioanalytical applications due to the financial, ethical and organizational issues involved in clinical trials. This could be accomplished due to the latest advances in modern analytical technology, particularly liquid chromatography–mass spectrometry. The present review discusses some of the emerging liquid chromatography–mass spectrometry technologies in improving DBS analysis for its innovative applications in the development of new drugs.     

Quantitative analysis of multiple fatty acid ethanolamides using ultra-performance liquid chromatography–tandem mass spectrometry

Fatty acid ethanolamides (FAE) represent a group of lipid signaling molecules associated with many physiological and pharmacological actions; however, low FAE tissue levels pose challenges in terms of analytical characterization. The objective was to develop a competent ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) method for analysis of multiple FAE in animal and human tissue samples. Analytes were extracted using lipid-phase and solid-phase extraction procedures. Chromatographic separation was achieved using a gradient elution in 8 min. FAE were quantified by MS/MS in positive electrospray ionization mode. Linearity was shown in lower and higher FAE concentration ranges, with a limit of quantification (LOQ) ≤0.2 ng/ml for FAE including alpha-linolenoylethanolamide (ALEA), arachidonoylethanolamide (AEA), docosahexaenoylethanolamide (DHEA), linoleoylethanolamide (LEA), oleoylethanolamide (OEA) and palmitoylethanolamide (PEA). Accuracy was shown to be between 92.4% and 108.8%, and precision was <10% for all FAE species. In sum, this sensitive and reproducible method can be used to simultaneously determine multiple FAE at low concentrations in order to facilitate further study of the role of FAE on physiological state.     

Absorption and metabolism of short‐chain fatty acids in ruminants

Short‐chain fatty acids (SCFA), viz. acetate, propionate and butyrate are quantitatively important substrates in ruminant energy metabolism. In the reviewed literature, 16–44% of ME intake was recovered as portal appearance of SCFA. This is considerably lower than expected when related to the estimated intra‐gastric flux of SCFA. The discrepancy is caused by portal drained viscera metabolism of arterially abundant metabolites e.g., acetate and the metabolism of acetate and butyrate to acetoacetate and D‐3‐hydroxybu‐tyrate in the absorptive epithelia. Even though considerable variations between experiments on acetate and propionate appearance are found, there seems to be a great deal of evidence that the proportion of gastroin‐testinally produced acetate and propionate absorbed to the portal blood is 50–75%. The portal recovery of butyrate has been found to be between 10 and 36% dependent on intraruminal infusion rate.

It is concluded that major parts of acetate and propionate are directly absorbed to the portal vein. The true absorption rate of acetate can only be estimated by taking the portal drained viscera metabolism of arterial actetate into account. Butyrate is generally found to have a low recovery in the portal vein, but the production of D‐3‐hydroxybutyrate seems to be underestimated in major parts of the literature. It is therefore necessary to measure portal appearance as well as portal drained viscera metabolism to assess the quantitative as well as the qualitative contribution of SCFA and SCFA metabolites to whole animal metabolism.     

Identification of YH439 and its metabolites in rat urine by gas chromatography–mass spectrometry and liquid chromatography–mass spectrometry

YH439 is a potential drug candidate for the treatment of various hepatic disorders. YH439 and its three metabolites have been identified in rat urine by liquid chromatography–mass spectrometry (LC–MS) and by gas chromatography (GC)–MS. Identification of YH439 and its metabolites was established by comparing their GC retention times and mass spectra with those of the synthesized authentic standards. Both electron impact- and positive chemical ionization MS have been evaluated. The metabolism study was performed in the rat using oral administration of the drug. A major metabolite (YH438) was identified as the N-dealkylation product of YH439. Other identified metabolites were caused by the loss of the methyl thiazolyl amine group (metabolite II) from YH439, the isopropyl hydrogen malonate group (metabolite IV) and the decarboxylated product (metabolite III) of metabolite II.     

Determination of mitoxantrone in rat plasma by liquid chromatography–tandem mass spectrometry method: Application to a pharmacokinetic study

A rapid, sensitive and specific high performance liquid chromatography–tandem mass spectrometric (HPLC–MS/MS) method has been developed for quantification of mitoxantrone in rat plasma. The analyte and palmatine (internal standard) were extracted from plasma samples with diethyl ether–dichloromethane (3:2, v/v) and separated on a C₁₈ column. The chromatographic separation was achieved within 2.5 min using methanol–10 mM ammonium acetate containing 0.1% acetic acid as the mobile phase at a flow rate of 0.2 mL/min. The method was linear over the range of 0.5–500 ng/mL. The lower limit of quantification (LLOQ) was 0.5 ng/mL. Finally, the method was successfully applied to a pharmacokinetic study of mitoxantrone in rats following intravenous administration.     

Determination of bicyclol in dog plasma using liquid chromatography–tandem mass spectrometry

A sensitive and accurate method for determination of bicyclol in dog plasma was developed. Thermo Scientific TSQ Quantum triple quadrupole system with multiple ion monitoring (MIM) positive scanning mode was applied. Bicyclol and DDB (IS) sodium adduct molecular ions were monitored at m/z 413 and m/z 441 in both Q1 and Q3, respectively. The collision energy in Q2 was set to 15 eV. Precipitation method was employed in the extraction of bicyclol and DDB from the biological matrix. The method was validated over 1–500 ng/mL for bicyclol. The recovery was 96.5–109.5%, and the limit of quantitation (LOQ) detection was 1 ng/mL for bicyclol. The intra- and inter-day precision of the method at three concentrations was 3.3–14.3% with accuracy of 99.9–109.0%. The method was successfully applied to bioequivalence studies of bicyclol controlled-release formulation to obtain the pharmacokinetic parameters.     

Quantitation of itraconazole in rat heparinized plasma by liquid chromatography–mass spectrometry

A liquid chromatographic–mass spectrometric (LC–MS) assay was developed and validated for the determination of itraconazole (ITZ) in rat heparinized plasma using reversed-phase HPLC combined with positive atmospheric pressure ionization (API) mass spectrometry. After protein precipitation of plasma samples (0.1 ml) with acetonitrile containing nefazodone as an internal standard (I.S.), a 50-μl aliquot of the supernatant was mixed with 100 μl of 10 mM ammonium formate (pH 4.0). An aliquot of 25 μl of the mixture was injected onto a BDS Hypersil C₁₈ column (50×2 mm; 3 μm) at a flow-rate of 0.3 ml/min. The mobile phase comprising of 10 mM ammonium formate (pH 4) and acetonitrile (60:40, v/v) was used in an isocratic condition, and ITZ was detected in single ion monitoring (SIM) mode. Standard curves were linear (r²≥0.994) over the concentration range of 4–1000 ng/ml. The mean predicted concentrations of the quality control (QC) samples deviated by less than 10% from the corresponding nominal values; the intra-assay and inter-assay precision of the assay were within 8% relative standard deviation. Both ITZ and I.S. were stable in the injection solvent at room temperature for at least 24 h. The extraction recovery of ITZ was 96%. The validated assay was applied to a pharmacokinetic study of ITZ in rats following administration of a single dose of itraconazole (15 mg/kg).     

Quantification of thyrotropin-releasing hormone by liquid chromatography–electrospray mass spectrometry

Thyrotropin-releasing hormone (TRH) is involved in a wide range of biological responses. It has a central role in the endocrine system and regulates several neurobiological activities. In the present study, a rapid, sensitive and selective liquid chromatography–mass spectrometry method for the identification and quantification of TRH has been developed. The methodology takes advantage of the specificity of the selected-ion monitoring acquisition mode with a limit of detection of 1 fmol. Furthermore, the MS/MS fragmentation pattern of TRH has been investigated to develop a selected reaction monitoring (SRM) method that allows the detection of a specific b2 product ion at m/z 249.1, corresponding to the N-terminus dipeptide pyroglutamic acid–histidine. The method has been tested on rat hypothalami to evaluate its suitability for the detection within very complex biological samples.     

Determination of haloacetic acids in human urine by headspace gas chromatography–mass spectrometry

Haloacetic acids (HAAs) are water disinfection byproducts (DBPs) formed by the reaction of chlorine oxidizing compounds with natural organic matter in water containing bromine. HAAs are second to trihalomethanes as the most commonly detected DBPs in surface drinking water and swimming pools. After oral exposure (drinking, showering, bathing and swimming), HAAs are rapidly absorbed from the gastrointestinal tract and excreted in urine. Typical methods used to determine these compounds in urine (mainly from rodents) only deal with one or two HAAs and their sensitivity is inadequate to determine HAA levels in human urine, even those manual sample preparation protocols which are complex, costly, and neither handy nor amenable to automation. In the present communication, we report on a sensitive and straightforward method to determine the nine HAAs in human urine using static headspace (HS) coupled with GC–MS. Important parameters controlling derivatisation and HS extraction were optimised to obtain the highest sensitivity: 120 μl of dimethylsulphate and 100 μl of tetrabutylammonium hydrogen sulphate (derivatisation regents) were selected, along with an excess of Na₂SO₄ (6 g per 12 ml of urine), an oven temperature of 70 °C and an equilibration time of 20 min. The method developed renders an efficient tool for the precise and sensitive determination of the nine HAAs in human urine (RSDs ranging from 6 to 11%, whereas LODs ranged from 0.01 to 0.1 μg/l). The method was applied in the determination of HAAs in urine from swimmers in an indoor swimming pool, as well as in that of non-swimmers. HAAs were not detected in the urine samples from non-swimmers and those of volunteers before their swims; therefore, the concentrations found after exposure were directly related to the swimming activity. The amounts of MCAA, DCAA and TCAA excreted from all swimmers are related to the highest levels in the swimming pool water.     

Determination of melatonin in Acyrthosiphon pisum aphids by liquid chromatography–tandem mass spectrometry

Melatonin is a hormone mainly involved in the regulation of circadian and seasonal rhythms in both invertebrates and vertebrates. Despite the identification of melatonin in many insects, its involvement in the insect seasonal response remains unclear. A liquid chromatography tandem mass spectrometry (LC–MS/MS) method has been developed for melatonin analysis in aphids (Acyrthosiphon pisum) for the first time. After comparing two different procedures and five extraction solvents, a sample preparation procedure with a mixture of methanol/water (50:50) was selected for melatonin extraction. The method was validated by analyzing melatonin recovery at three spiked concentrations (5, 50 and 100 pg/mg) and showed satisfactory recoveries (75–110%), and good repeatability, expressed as relative standard deviation (<10%). Limits of detection (LOD) and quantitation (LOQ) were 1 pg/mg and 5 pg/mg, respectively. Eight concentration levels were used for constructing the calibration curves which showed good linearity between LOQ and 200 times LOQ. The validated method was successfully applied to 26 aphid samples demonstrating its usefulness for melatonin determination in insects. This is -to our knowledge- the first identification of melatonin in aphids by LC–MS/MS.     

Determination of picamilon concentration in human plasma by liquid chromatography–tandem mass spectrometry

A rapid liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed and validated for the determination of picamilon concentration in human plasma. Picamilon was extracted from human plasma by protein precipitation. High performance liquid chromatography separation was performed on a Venusil ASB C₁₈ column with a mobile phase consisting of methanol ?10 mM ammonium acetate–formic acid (55:45:01, v/v/v) at a flow rate of 0.65 ml/min. Acquisition of mass spectrometric data was performed in selected reaction monitoring mode, using the transitions of m/z 209.0 → m/z (78.0 + 106.0) for picamilon and m/z 152.0 → m/z (93.0 + 110.0) for paracetamol (internal standard). The method was linear in the concentration range of 1.00–5000 ng/ml for the analyte. The lower limit of quantification was 1.00 ng/ml. The intra- and inter-assay precision were below 13.5%, and the accuracy was between 99.6% and 101.6%. The method was successfully applied to characterize the pharmacokinetic profiles of picamilon in healthy volunteers. This validated LC–MS/MS method was selective and rapid, and is suitable for the pharmacokinetic study of picamilon in humans.     

Using liquid chromatography–tandem mass spectrometry to quantify monohydroxylated metabolites of polycyclic aromatic hydrocarbons in urine

We present an assay which employs enzyme digestion and solid phase extraction followed by liquid chromatography–tandem mass spectrometry to simultaneously quantify 16 hydroxylated polycyclic aromatic hydrocarbons (OHPAHs) in 3-ml samples of urine. The analytes consisted of 2-, 3-, and 4-ring OHPAHs, namely, 1- and 2-hydroxynaphthalene (1- and 2-OHNAP), 2-hydroxyfluorine (2-OHFLU), 1-, 2-, 3-, 4-, and 9-hydroxyphenanthrene (1-, 2-, 3-, 4-, and 9-OHPHE), 1-hydroxypyrene (1-OHPYR), 1- and 2-hydroxybenzo(a)anthracene (1- and 2-OHBAA), 3- and 6-hydroxychrysene (3- and 6-OHCHR) and 3-, 7-, and 9-hydroxybenzo(a)pyrene (3-, 7-, and 9-OHBAP). The method was validated using urine samples from steel workers and control subjects. The coefficients of variation of the method for the particular analytes were between 7% and 27% and the limits of quantitation were between 0.002 and 0.010 μg/l urine. The 2- and 3-ring OHPAHs were easily quantified in all subjects. However, 1-OHPYR was the only representative of the 4- and 5-ring metabolites that could be quantified. Pairwise correlations showed that all OHPAHs were highly correlated with each other (0.553 ≤ r ≤ 0.910) and with 1-OHPYR (0.614 ≤ r ≤ 0.910), the metabolite most widely accepted as a short-term biomarker of exposure to PAHs. The analyte, 2-OHNAP exhibited the lowest pairwise correlations with the other OHPAHs (0.542 ≤ r ≤ 0.628), presumably due to confounding by smoking. Metabolites of phenanthrene, an abundant PAH and the smallest to possess a bay region, are promising OHPAHs for characterizing both exposures to PAHs and the various metabolic pathways.     

Determination of cymipristone in human plasma by liquid chromatography–electrospray ionization-tandem mass spectrometry

A rapid, specific and sensitive liquid chromatography–electrospray ionization-tandem mass spectrometry method was developed and validated for determination of cymipristone in human plasma. Mifepristone was used as the internal standard (IS). Plasma samples were deproteinized using methanol. The compounds were separated on a ZORBAX SB C₁₈ column (50 mm × 2.1 mm i.d., dp 1.8 μm) with gradient elution at a flow-rate of 0.3 ml/min. The mobile phase consisted of 10 mM ammonium acetate and acetonitrile. The detection was performed on a triple-quadruple tandem mass spectrometer by selective reaction monitoring (SRM) mode via electrospray ionization. Target ions were monitored at [M+H]⁺ m/z 498 → 416 and 430 → 372 in positive electrospray ionization (ESI) mode for cymipristone and IS, respectively. Linearity was established for the range of concentrations 0.5–100 ng/ml with a coefficient correlation (r) of 0.9996. The lower limit of quantification (LLOQ) was identifiable and reproducible at 0.5 ng/ml. The validated method was successfully applied to study the pharmacokinetics of cymipristone in healthy Chinese female subjects.     

A quantitative headspace–solid-phase microextraction–gas chromatography–flame ionization detector method to analyze short chain free fatty acids in rat feces

This study sought to develop and validate a quantitative method to analyze short chain free fatty acids (SCFAs) in rat feces by solid-phase microextraction and gas chromatography (SPME–GC) using the salt mixture ammonium sulfate and sodium dihydrogen phosphate as salting out agent. Conditioning and extraction time, linearity, limits of detection and quantification, repeatability, and recovery were evaluated. The proposed method allows quantification with improved sensitivity as compared with other methods exploiting SPME–GC. The method has been applied to analyze rat fecal samples, quantifying acetic, propionic, isobutyric, butyric, isopentanoic, pentanoic, and hexanoic acids.     

Simultaneous measurement of aldosterone and cortisol by high-performance liquid chromatography–tandem mass spectrometry: Application to dehydration–rehydration studies

Aldosterone and cortisol are useful biomarkers of dehydration and stress, respectively. The aim of this study was to develop an HPLC–tandem mass spectrometric method for the simultaneous measurement of aldosterone and cortisol in human plasma that could be applied to the study of athletes undergoing exercise and rehydration. Samples were prepared and analysed using an on-line sample preparation/HPLC system coupled to a triple quadrupole tandem-mass spectrometer. Samples (200 μL) were pre-treated and extracted on Hysphere C18 HD cartridges (7 μm, Spark Holland). Chromatography was performed on a Sunfire C18 analytical column (50 mm × 3.0 mm, 3 μm, Waters) under isocratic conditions at a flow rate of 0.3 mL/min. The mobile phase consisted of 35% acetonitrile/water. Mass spectrometric detection was by selected reaction monitoring using negative electrospray ionization conditions. The assay had an analytical range of 25–500 pg/mL and 25–500 ng/mL for aldosterone and cortisol, respectively (r² > 0.992, n = 22). Inter-day accuracy and imprecision for quality control samples was 99.4–106% and <16%, respectively (n = 10). In a study of nine human subjects, both aldosterone and cortisol concentrations reflected the expected physiological responses to dehydration, rehydration and exercise when measured by this method. The reported method is suitable to facilitate the study of athletes undergoing dehydration and rehydration protocols.