Simultaneous quantification of ten Amadori compounds in tobacco using liquid chromatography with tandem mass spectrometry

Amadori compounds are aroma precursors formed in the initial phase of the Maillard reaction. Based on their similar structures, simultaneous quantification of more than six Amadori compounds in tobacco has not been reported yet. In this study, a simple and rapid method was developed to simultaneously quantify ten Amadori compounds including the isomers of Fructose‐isoleucine and Fructose‐leucine in tobacco. The separation was performed on an Atlantis T₃ column (2.1 × 250 mm, 5 μm) by gradient elution using acetonitrile and water as the mobile phases. The quantification method was systematically evaluated and proven to be sensitive and accurate. The linearity was good, with correlation coefficients of 0.9977–0.9999. The limits of detection and quantitation were 1.354–2.532 and 4.516–8.444 ng/mL, respectively. The recoveries were 84.0–119.6%, and the relative standard deviations were 1.33–5.40%. The method was used to analyze the changes in the amounts of ten Amadori compounds in tobacco before and after tobacco primary processing. The analysis shows that the Maillard reaction occurs during the short processing period.     

Investigations on the degradation of aspartame using high-performance liquid chromatography/tandem mass spectrometry

Aspartame is a widely used sweetener,the long-term safety of which has been controversial ever since it was accepted for human consumption.It is unstable and can produce some harmful degradation products under certain storage conditions.A high-performance liquid chromatography/tandem mass spectrometry method was developed for the simultaneous analysis of aspartame and its four degradation products,including aspartic acid,phenylalanine,aspartyl-phenylalanine and 5-benzyl-3,6-dioxo-2-piperazieacetic acid in water and in diet soft drinks.Aspartame and its four degradation products were quantified by a matrix matched external standard calibration curve with excellent correlation coefficients.The limits of detection were 0.16–5.8 mg/L,which exhibited higher sensitivity than common methods.This method was rapid,sensitive,specific and capable of eliminating matrix interferences.It was also applied to the study of the degradation of aspartame at various pH and temperatures.The results indicated that aspartame was partly degraded under strong acidic or basic conditions and the extent of degradation increased with increasing temperature.     

Simultaneous quantification of oleuropein and its metabolites in rat plasma by liquid chromatography electrospray ionization tandem mass spectrometry

Oleuropein (OE) is the cardinal bioactive compound derived from Olea europaea and possesses numerous beneficial properties for human health. However, despite the plethora of analytical methods that have studied the biological fate of olive oil‐derived bioactive compounds, no validated methodology has been published to date for the simultaneous determination of OE, along with all its major metabolites. In this study, a liquid chromatography‐electrospray ionization‐tandem mass spectrometry (LC‐ESI MS/MS) method has been developed and validated for the quantification of OE, simultaneously with its main metabolites hydroxytyrosol, 2‐(3,4‐dihydroxyphenyl)acetic acid, 4‐(2‐hydroxyethyl)‐2‐methoxy‐phenol or homovanillyl alcohol, 2‐(4‐hydroxy‐3‐methoxyphenyl)acetic acid or homovanillic acid, and elenolic acid in rat plasma matrix. Samples were analyzed by LC‐ESI MS/MS prior to and after enzymatic treatment. A solid‐phase extraction step with high mean recovery for all compounds was performed as sample pretreatment. Calibration curves were linear for all bioactive compounds over the range studied, while the method exhibited good accuracy, intra‐ and inter‐day precision. The limit of detection was in the picogram range (per milliliterof plasma) for HT and OE and in the nanogram range (per milliliter of plasma) for the other analytes, and the method was simple and rapid. The developed methodology was successfully applied for the simultaneous quantification of OE and its aforementioned metabolites in rat plasma samples, thus demonstrating its suitability for pharmacokinetics, as well as bioavailability and metabolism studies. Copyright © 2009 John Wiley & Sons, Ltd.     

Combination of liquid chromatography/tandem mass spectrometry and gas chromatography/mass spectrometry for the detection of 21 anabolic steroid residues in bovine urine

For the detection of anabolic steroid residues in bovine urine, a highly sensitive liquid chromatographic/electrospray ionization tandem mass spectrometric (LC/ESI-MS/MS) method was developed using both positive and negative ionization. For four compounds the ESI mode was not sensitive enough and gas chromatographic/mass spectrometric GC/MS detection was therefore still necessary as a complementary method. The sample clean-up consisted of solid-phase extraction (SPE) on a C(18) column followed by enzymatic hydrolysis and a second solid-phase extraction on a combination of a C(18) and a NH(2) column. After this last SPE clean-up, the eluate was split into two equal aliquots. One aliquot was further purified and after derivatization used for GC/MS analysis. The other aliquot was analyzed with LC/MS/MS in both ESI+ and ESI- modes. The method was validated according to the European Commission Decision 2002/657/EC. Decision limits (CCalpha) were between 0.16 and 1 ng ml(-1) for the compounds detected with the LC/MS/MS method. The developed method is used in routine analysis in our laboratory.     

Nevirapine quantification in human plasma by high-performance liquid chromatography coupled to electrospray tandem mass spectrometry. Application to bioequivalence study

A rapid, sensitive and specific method to quantify nevirapine in human plasma using dibenzepine as the internal standard (IS) was developed and validated. The method employed a liquid-liquid extraction. The analyte and the IS were chromatographed on a C(18) analytical column, (150 x 4.6 mm i.d. 4 microm) and analyzed by tandem mass spectrometry in the multiple reaction monitoring mode. The method had a chromatographic run time of 5.0 min and a linear calibration curve over the range 10-5000 ng ml(-1) (r(2) > 0.9970). The between-run precision, based on the relative standard deviation for replicate quality controls was 1.3% (30 ng ml(-1)), 2.8% (300 ng ml(-1)) and 3.6% (3000 ng ml(-1)). The between-run accuracy was 4.0, 7.0 and 6.2% for the above-mentioned concentrations, respectively. This method was employed in a bioequivalence study of two nevirapine tablet formulations (Nevirapina from Far-Manguinhos, Brazil, as a test formulation, and Viramune from Boehringer Ingelheim do Brasil Química e Farmacêutica, as a reference formulation) in 25 healthy volunteers of both sexes who received a single 200 mg dose of each formulation. The study was conducted using an open, randomized, two-period crossover design with a 3 week washout interval. The 90% confidence interval (CI) of the individual ratio geometric mean for Nevirapina/Viramune was 96.4-104.5% for AUC((0-last)), 91.4-105.1% for AUC((0-infinity)) and 95.3-111.6% for C(max) (AUC = area under the curve; C(max) = peak plasma concentration). Since both 90% CI for AUC((0-last)) and AUC((0-infinity)) and C(max) were included in the 80-125% interval proposed by the US Food and Drug Administration, Nevirapina was considered bioequivalent to Viramune according to both the rate and extent of absorption.     

Determination of spinetoram in leafy vegetable crops using liquid chromatography and confirmation via tandem mass spectrometry

Spinetoram is a second-generation member of the spinosyn class, all members of which have been shown to be effective in insect control via a novel mode of action. Spinetoram is a mixture of 3'-O-ethyl-5, 6-dihydro spinosyn J (XDE-175-J) and 3'-O-ethyl spinosyn L (XDE-175-L). In order to establish a determination method for the analysis of spinetoram residues in crops, commercial product (5% suspension concentrate spinetoram) was applied to two leafy vegetables (Garland chrysanthemum and Aster scaber) on different spraying schedules. The analytical method used herein was based on a reversed-phase separation on a C(18) column, isocratic elution and UV detection. The analytes were confirmed via tandem mass spectrometry. The method was linear over a concentration range of 0.05-10 ppm with a correlation coefficient in excess of 0.9998. The recoveries of XDE-175-J and XDE-175-L from the two vegetables ranged between 86.04 and 98.87% at spiking levels of 1 and 5 ppm. The relative standard deviations were no more than 7% for all recovery tests conducted herein. The calculated limits of detection and quantification were 0.01 and 0.03 ppm for both XDE-175-J and XDE-175-L. The levels of residues in two vegetables treated under a fixed schedule in the greenhouse were 6.21-0.55 ppm (maximum residue limit (MRL) = 7 ppm). In sum, this method constitutes an easy and reliable technique for the determination of spinetoram in leafy vegetables.     

Differentiation of betamethasone and dexamethasone using liquid chromatography/positive electrospray tandem mass spectrometry and multivariate statistical analysis

Betamethasone and dexamethasone are two corticosteroids differing in the stereoisomery of their C-16 methyl group. These two compounds are imperfectly separated by reversed-phase liquid chromatography and their mass spectra are very similar, leading to a difficult unambiguous identification according to European criteria. A method is proposed for differentiating betamethasone and dexamethasone using liquid chromatography/tandem mass spectrometry and multivariate statistical analysis. Multiple analysis of variance was used for the justification and the selection of diagnostic ions. Principal component analysis permitted the suitability of the approach to be tested on a large number of samples. Discriminant factorial analysis was finally performed to build a decisional model based on the six most significant ions. This novel utilization of mass spectrometric data appeared efficient for the unambiguous identification of the target analytes in urine samples.     

Simultaneous quantification of cilostazol and its primary metabolite 3,4-dehydrocilostazol in human plasma by rapid liquid chromatography/tandem mass spectrometry

A simple, rapid, sensitive and selective liquid chromatography/electrospray tandem mass spectrometry method was developed and validated for the simultaneous quantification of cilostazol and its primary metabolite 3,4-dehydrocilostazol in human plasma using mosapride as an internal standard. The method involves a simple one-step liquid-liquid extraction with a diethyl ether and dichloromethane mixture (7:3). The analytes were chromatographed using an isocratic mobile phase on a reversed-phase C₁₈ column and analyzed by mass spectrometry in the multiple reaction monitoring mode using the respective [M+H]⁺ ions, m/z 370/288 for cilostazol, m/z 368/286 for 3,4-dehydrocilostazol and m/z 422/198 for the internal standard. The assay exhibited a linear dynamic range of 5–2,000 ng/mL for cilostazol and 5–400 ng/mL for 3,4-dehydrocilostazol in human plasma. The lower limit of quantitation was 5 ng/mL for both cilostazol and its metabolite. Acceptable precision and accuracy were obtained for concentrations over the standard curve ranges. A run time of 2.5 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 pharmacokinetics, bioavailability or bioequivalence studies.      

Simultaneous determination of metabolites of trimethylbenzenes,dimethylbenzylmercapturicacid and dimethylhippuric acid,in human urine by solid-phase extraction followed by liquid chromatography tandem mass spectrometry

We describe a novel method for the determination of three kinds of dimethylbenzylmercapturic acids (DMM) and six kinds of dimethylhippuric acids (DMH), found in urine as metabolites of trimethylbenzenes, based on liquid chromatography/electrospray ionization tandem mass spectrometry. A solid-phase extraction procedure was used for the extractions of DMM and DMH from a urine sample, and the separation was performed on a reversed-phase C(30) column. The analytes were ionized by electrospray in the positive-ion mode. Operating in the multiple reaction monitoring mode, the linearity of the relative mass spectrometric responses to the internal standard versus analyte concentrations were established in the range 0.1-100 ng ml(-1). The extraction procedure was rapid and the relative standard deviations were below 5%. The detection limits of DMM and DMH in the urine by the proposed method were in the ranges 0.26-0.41 and 0.42-2.0 ng l(-1), respectively. Furthermore, DMM and DMH were detected in a urine sample from an individual who did not suffer from occupational exposure to trimethylbenzenes, by using this method.     

Simultaneous determination of Delta9-tetrahydrocannabinol, 11-hydroxy-Delta9-tetrahydrocannabinol and 11-nor-9-carboxy- Delta9-tetrahydrocannabinol in human plasma by high-performance liquid chromatography/tandem mass spectrometry

A rapid and sensitive method for the simultaneous confirmatory analysis of three forensic most relevant cannabinoids, Delta(9)-tetrahydrocannabinol (THC), 11-hydroxy-Delta(9)-tetrahydrocannabinol (11-OH-THC) and 11-nor-9-carboxy-Delta(9)-tetrahydrocannabinol (THC-COOH), by means of high-performance liquid chromatography/tandem mass spectrometry (LC/MS/MS) in human plasma was developed and fully validated. Sample clean-up was performed by automated silica-based solid-phase extraction and the separation was carried out using a PhenylHexyl column (50 x 2 mm i.d., 3 micro m) and acetonitrile-5 mM ammonium acetate gradient elution. Data were acquired with an API 3000 LC/MS/MS system equipped with a turboionspray interface and triple quadrupole mass analyzer using positive electrospray ionization and multiple reaction monitoring. Two MS/MS transitions for each substance were monitored and deuterated analogues of analytes were used as internal standards for quantitation. The limit of quantitation was 0.8 ng ml(-1) for THC, 0.8 ng ml(-1) for 11-OH-THC and 4.3 ng ml(-1) for THC-COOH and linearity with a correlation coefficient r(2) = 0.999 was achieved up to 100 ng ml(-1) for THC and 11-OH-THC and 500 ng ml(-1) for THC-COOH. The limits of detection were 0.2 ng ml(-1) for THC, 0.2 ng ml(-1) for 11-OH-THC and 1.6 ng ml(-1) for THC-COOH. The developed LC/MS/MS method was also successfully used for the determination of THC-COOH-glucuronide, the phase II metabolite of THC-COOH.     

Determination of paraquat and diquat in human body fluids by high-performance liquid chromatography/tandem mass spectrometry

Paraquat (PQ) and diquat (DQ) in human whole blood and urine were analyzed by high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) with positive ion electrospray ionization (ESI). The compounds were extracted with Sep-Pak C18 cartridges from whole blood and urine samples containing ethyl paraquat as an internal standard. The separation of PQ and DQ was carried out using ion-pair chromatography with heptafluorobutyric acid in 20 mM ammonium acetate and acetonitrile gradient elution for successful coupling with MS. Both compounds formed base peaks due to [M-H]+ ions by HPLC/ESI-MS and the product ions produced from each [M-H]+ ion by HPLC/MS/MS. Selective reaction monitoring (SRM) showed much higher sensitivity for both body fluids. Therefore, a detailed procedure for the detection of compounds by SRM with HPLC/MS/MS was established and carefully validated. The recoveries of PQ and DQ were 80.8-95.4% for whole blood and 84.2-96.7% for urine. The calibration curves for PQ and DQ showed excellent linearity in the range of 25-400 ng ml(-1) of whole blood and urine. The detection limits were 10 ng ml(-1) for PQ and 5 ng ml(-1) for DQ in both body fluids. The intra- and inter-day precision for both compounds in whole blood and urine samples were not greater than 13.0%. The data obtained from the determination of PQ and DQ in rat blood after oral administration of the compounds are also presented.     

Simultaneous quantification of N‐butylthiophosphoric triamide and dicyandiamide in urea formulation by liquid chromatography with tandem mass spectrometry

A new liquid chromatography with tandem mass spectrometry method employing a mixed‐mode zwitterionic stationary phase was developed for simultaneous determination of urease inhibitor (N‐butylthiophosphoric triamide) and nitrification inhibitor (dicyandiamide) in urea fertilizer. Molecular modeling based on density functional theory calculations was employed to provide an insight into the interaction mechanism of urea, dicyandiamide, and N‐butylthiophosphoric triamide with zwitterionic stationary phase in chromatographic separation system. The detection of analytes was performed on a triple quadrupole tandem mass spectrometer in multiple reaction monitoring mode using positive electrospray ionization. The ion transitions monitored were m/z 85→68 for dicyandiamide and m/z 168.2→74 for N‐butylthiophosphoric triamide, respectively. The standard calibration curves of dicyandiamide and N‐butylthiophosphoric triamide were linear over the range of 1.0 ? 15 ppm (coefficient of determination = 0.9984), 0.05 ? 1 ppm (coefficient of determination = 0.9995), with limit of detection of 25 and 5 ppb, respectively. The recoveries of low, middle, and high concentrations were from 96.7 to 105.8% for N‐butylthiophosphoric triamide and 94.4 to 105.8% for dicyandiamide with accuracy (relative error %) of ≤5.8% and ≤5.8%, the precision (coefficients of variation) was ≤2.0% and ≤2.9%, respectively. The validated method was successfully applied on real urea samples to determine N‐butylthiophosphoric triamide and dicyandiamide simultaneously.     

Sensitive liquid chromatography tandem mass spectrometry method for the quantification of Quetiapine in plasma

A sensitive high-performance liquid chromatography-tandem mass spectrometry method was developed and validated for the quantification of quetiapine in rat plasma. Following liquid-liquid extraction, the analyte was separated using a gradient 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 384 to m/z 221 for quetiapine and m/z 327 to m/z 270 for the internal standard. The assay exhibited a linear dynamic range of 0.25-500 ng/mL for quetiapine in rat plasma. The lower limit of quantification was 0.25 ng/mL with a relative standard deviation of less than 7%. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. The validated method was successfully used to analyze rat plasma samples for application in pre-clinical pharmacokinetic studies. This method in rodent plasma could be adapted for quetiapine assay in human plasma.     

Simultaneous pharmacokinetics and stability studies of physalins in rat plasma and intestinal bacteria culture media using liquid chromatography with mass spectrometry

Physalins are the major steroidal constituent of Physalis plants and display a range of biological activities. For this study, a rapid and sensitive high‐performance liquid chromatography with triple quadrupole mass spectrometry method was developed for the simultaneous quantification of six physalins. Specifically, it was for the quantification of physalin A, physalin B, physalin D, physalin G, 4,7‐didehydroneophysalin B, and isophysalin B in rat plasma and rat intestinal bacteria. After a solid‐phase extraction, analytes and internal standards (prednisolone) were separated on a Shield reverse‐phase C18 column (measuring 3 mm × 150 mm with an internal diameter of 3.5 μm) and determined using multiple reactions in a monitoring mode with a positive‐ion electrospray ionization source. The mobile phase was a mixture of 0.1% formic acid in water (A) and acetonitrile (B) and was used at a flow rate of 0.6 mL/min. The intra‐ and interday precisions were within 15% with accuracies ranging from 86.2 to 114%. The method was validated and successfully applied to pharmacokinetics and stability studies of six physalins in rat plasma and rat intestinal bacteria, respectively. The results showed that physalin B and isophysalin B could not be absorbed by rats, and rat intestinal bacteria could quickly transform physalins.     

Liquid chromatography tandem mass spectrometry method for the quantification of amisulpride with LLOQ of 100 pg/mL using 100 microL of plasma

A sensitive and selective high-performance liquid chromatography-positive ion electrospray tandem mass spectrometry method was developed and validated for the quantification of amisulpride in 100 microL of human plasma. Following liquid-liquid 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 370-242 for amisulpride and m/z 341-112 for the internal standard. The assay exhibited a linear dynamic range with a lower range of 0.1-100 ng/mL and a higher range of 1-500 ng/mL of amisulpride in human plasma. The lower limit of quantification was 0.1 ng/mL with a relative standard deviation of less than 10%. Acceptable precision and accuracy were obtained for both linearity ranges. A run time of 2.0 min for each sample made it possible to analyze more than 275 human plasma samples per day. The validated method has been successfully used to analyze plasma samples for application in pharmacokinetic studies.     

Dissipation dynamics and final residues of flutriafol in tobacco and soil using ultrasound-assisted extraction before liquid chromatography/tandem mass spectrometry

The dissipation dynamics and final residues of flutriafol on tobacco plant and soil were studied under field conditions. The residues of flutriafol in soil, green tobacco leaves and cured tobacco leaves were extracted by ultrasound-assisted extraction, cleaned up by dispersive solid-phase extraction and detected by liquid chromatography with tandem mass spectrometry. The limits of detection of flutriafol in soil, green tobacco leaves and cured tobacco leaves were 0.006, 0.033 and 0.033 mg·kg^?1, respectively. The limits of quantification of flutriafol in soil, green tobacco leaves and cured tobacco leaves were 0.02, 0.1 and 0.1 mg·kg^?1, respectively. Recoveries were 72.9–102% with relative standard deviations of less than 12% in soil and tobacco matrix. For field experiments, the half-lives of flutriafol in soil and green tobacco leaves were 9.2–11.5 and 9.5–11.1 days, respectively. At harvest, the final residue levels of flutriafol in cured tobacco leaves collected 21 days after one application at the recommended dosage were below 2.0 mg/kg. The maximum residue limit maximum residue limit (MRL) for flutriafol in tobacco has not yet been established in any countries. The data could help the Chinese Government to establish the MRL of flutriafol in tobacco and provide guidance on the proper use of flutriafol.     

Selective enrichment and quantification of okadaic acid in shellfish using an immunomagnetic‐bead‐based liquid chromatography with tandem mass spectrometry assay

Okadaic acid is a marine biotoxin that primarily occurs in shellfish and can cause diarrheic shellfish poisoning in humans. When analyzing biological samples using liquid chromatography with tandem mass spectrometry, the presence of complex matrices is a major issue. Thus, it is crucial to selectively and simply extract the target analyte from samples and minimize matrix effects simultaneously. To meet this need, an immunomagnetic‐bead‐based liquid chromatography with tandem mass spectrometry method was developed to detect okadaic acid in shellfish. Magnetic beads bound to monoclonal antibody against okadaic acid were used as affinity probes to specifically enrich okadaic acid in samples, which effectively eliminated matrix effects. A magnetic separator was used to aggregate and separate magnetic particles from sample matrices, and methanol was used to elute okadaic acid from the magnetic beads. Standard solution prepared with methanol was employed directly for quantitative analysis. Several experimental conditions were optimized to improve performance. The method is of interest as a rapid (10 min) sample clean‐up and selective enrichment tool, and it showed good linearity and sensitivity, with reported limits of detection and quantitation of 3 and 10 μg/kg, respectively. Fifty‐three shellfish samples from an aquatic products market were tested using this method, and four samples positive for okadaic acid were found.     

Simultaneous analysis of indaziflam and its metabolites in pitaya using dispersive solid phase extraction coupled with liquid chromatography coupled with tandem mass spectrometry

A simple and efficient multiresidue method using dispersive solid phase extraction and liquid chromatography coupled with tandem mass spectrometry was developed for the targeted analysis of indaziflam and its five metabolites (indaziflam‐diaminotriazine, indaziflam‐carboxylic acid, indaziflam‐triazine indanone, indaziflam‐hydroxyethyl, and indaziflam‐olefin) in pitaya samples (including roots, plants, flowers, peels, pulp, and whole fruit). The analytes were extracted with acetonitrile, and the extracts were purified using multiwalled carbon nanotubes. The method was validated using pitaya samples spiked at 0.5, 5, and 50 µg/kg, and the average recoveries varied from 61.1 to 103.7% with relative standard deviations lower than 12.7% (n = 5). This method exhibited sufficient linearity within the concentration range of 0.1–100 µg/L. The limits of detection and quantification were in the ranges of 0.001–0.1 and 0.003–0.3 µg/kg, respectively. The method was successfully applied to analyze pitaya samples in Nanning, and no indaziflam or its metabolites were detected in the samples analyzed.     

液相色谱-串联质谱法检测贝类产品中的原多甲藻酸贝类毒素

建立了一种贝类组织中原多甲藻酸(azaspiracid, AZA)贝类毒素主要成分AZA1的高效液相色谱-串联质谱检测方法。本方法采用甲醇-水(80:20, v/v)溶液对贝类组织中AZA1进行提取,并用MAX阴离子交换固相萃取(SPE)柱富集净化,使用Atlantis dC18(150 mm×4.6 mm, 5.0 μm)色谱柱分离,以含有50 mmol/L甲酸和2 mmol/L甲酸铵的乙腈-水溶液(80:20, v/v)为流动相进行等度洗脱,质谱采用选择反应监测(SRM)模式。AZA1在5 min内获得完全分离,且在48.85～2 442 ng/L范围内线性良好,相关系数为0.998 1。该方法检出限(S/N=3)为11.00 pg/g,添加水平为36.64、73.27、146.54 pg/g时的平均回收率为75.8%～82.5%(n=6),相对标准偏差小于10%。利用该方法对采自大连、青岛、广州水产品市场上的112个贝类样品进行了分析,发现采自大连和广州的部分贝类样品中含有AZA1。结果表明,该方法具有简单、快速、灵敏度高等特点,能充分满足贝类中AZA1检测的要求。     

Quantitative analysis of 5 alpha-reductase inhibitors in DU145 cells using matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry and high-performance liquid chromatography/tandem mass spectrometry

N-(Dicyclohexyl)acetylpiperidine-4-benzylidene-4-carboxylic acid (1) is an excellent in vitro inhibitor of 5 alpha-reductase (5 alpha R). Compound 1 showed, however, much lower inhibition activity of 5 alpha R in vivo than in vitro, which might be caused by poor membrane permeability. The methyl ester of 1 (1a) was therefore tested as a model prodrug to see if it has better permeability properties than the corresponding acid 1. It was also monitored that this methyl ester was cleaved into the active compound 1 within the DU145 cells. Quantitative matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) and high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) methods were established with reliable linearity factors (0.996 for MALDI-TOFMS and 0.998 for HPLC/MS/MS) and reproducibility (relative standard deviation = 6.5% for MALDI-TOFMS and 2.8% for HPLC/MS/MS). The samples for MS analysis were effectively prepared from the cell homogenates using solid-phase extraction, with a high recovery of 90% on average. The intracellular amount of 1a (1.7 nmol) was much higher than that of 1 (0.032 nmol) in DU145 cells after 6 h of incubation. After incubation with the ester (1a), the cleaved acid (1) was detected within the cells. The concentration of acid 1 (0.045 nmol) in this experiment was higher than the acid content (0.032 nmol) after direct incubation with 1. Surprisingly, high amounts of the cleaved compound 1 were found outside the cells after 6 h of incubation with 1a.