Quantification of artemisinin in human plasma using liquid chromatography coupled to tandem mass spectrometry

A liquid chromatographic tandem mass spectroscopy method for the quantification of artemisinin in human heparinised plasma has been developed and validated. The method uses Oasis HLB™ μ-elution solid phase extraction 96-well plates to facilitate a high throughput of 192 samples a day. Artesunate (internal standard) in a plasma–water solution was added to plasma (50 μL) before solid phase extraction. Artemisinin and its internal standard artesunate were analysed by liquid chromatography and MS/MS detection on a Hypersil Gold C18 (100 mm × 2.1 mm, 5 μm) column using a mobile phase containing acetonitrile–ammonium acetate 10 mM pH 3.5 (50:50, v/v) at a flow rate of 0.5 mL/min. The method has been validated according to published FDA guidelines and showed excellent performance. The within-day, between-day and total precisions expressed as R.S.D., were lower than 8% at all tested quality control levels including the upper and lower limit of quantification. The limit of detection was 0.257 ng/mL for artemisinin and the calibration range was 1.03–762 ng/mL using 50 μL plasma. The method was free from matrix effects as demonstrated both graphically and quantitatively.     

Simultaneous determination of the flavonoids robinin and kaempferol in human breast cancer cells by liquid chromatography-tandem mass spectrometry

An accurate, precise and sensitive method was developed and validated for the simultaneous quantification of the flavonoid glycoside robinin, and its algycone kaempferol in human breast cancer MCF-7 cells. The application of liquid chromatography-tandem mass spectrometry (LC/MS/MS) with a TurboIonspray interface in negative mode under multiple reactions monitoring was investigated. Chromatographic separation was achieved on a C(18) column using a mobile phase consisting of (A) water with 0.025% formic acid and 1mM ammonium formate and (B) acetonitrile with 0.025% formic acid. Rutin was used as the internal standard for robinin and fisetin as the internal standard for kaempferol. The assay had a limit of detection of 0.1ng/ml for both compounds when present in cell lysate. The calibration curves were linear from 1 to 250ng/ml (r>0.999) for each compound. The intra- and inter-day coefficients of variation were less than 10% and intra- and inter-day accuracies were within 11%. This assay was successfully applied in a robinin cellular uptake study to determine the intracellular concentrations of robinin in MCF-7 cells.     

A rapid, simple, specific liquid chromatographic-electrospray mass spectrometry method for the determination of finasteride in human plasma and its application to pharmacokinetic study

A fast, accurate, sensitive, selective and reliable method using reversed-phase high-performance liquid chromatography-mass spectrometry coupling with an electrospray ionization interface was developed and validated for the determination of finasteride in human plasma. After deprotienation with acetonitrile, centrifugation, evaporation to dryness and dissolving in mobile phase, satisfactory separation was achieved on a Hypersil-Keystone C(18) reversed-phase column using a mobile phase consisting of acetonitrile-water (46:54, v/v), 0.1% acetic acid and 0.1% trifluoracetic acid. Carbamazepine (IS) was used as internal standard. This method involved the use of the [M+H](+) ions of finasteride and IS at m/z 373 and 237 with the selective ion monitoring (SIM) mode. The calibration curve was linear in the range of 0.2-120 ng ml(-1). The limit of quantification for finasteride in plasma was 0.2 ng ml(-1) with good accuracy and precision. The intra-assay precision and accuracy were in the range of 2.1-11.2% and -1.3% to 8.5%, respectively. The inter-assay precision and accuracy were in the order of 3.4-12.1% and -1.5% to 11.5%, respectively. The mean sample extract recoveries of the method were higher than 85% and 74% for finasteride and internal standard (IS), respectively. The assay has been successfully used to estimate the pharmacokinetics of finasteride after oral administration of a 5mg tablet of finasteride to 24 healthy volunteers.     

Sensitive and selective liquid chromatography-tandem mass spectrometry method for the determination of five ganoderic acids in Ganoderma lucidum and its related species

The present paper describes a novel, sensitive and selective liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous analysis of ganoderic acids C(2), B, A, H, D in Ganoderma lucidum and its related species. Ganoderma samples were prepared using simple ultrasonic extraction. Chromatographic separation was carried out on an Agilent Zorbax XDB C(18) column (250 mm × 4.6 mm i.d., 5μm) with an isocratic mobile phase consisting of acetonitrile, water and formic acid (42:58:0.5, v/v/v). Mass spectrometric detection was achieved by a triple-quadrupole mass spectrometer equipped with an atmospheric pressure chemical ionization (APCI) interface operating in negative and positive ionization mode via a single within-run polarity switching. Quantitation of five ganoderic acids was performed using selective reaction monitoring (SRM) mode. The intra- and inter-day precision was less than 6.2% and the accuracy ranged from 90.0% to 105.7%. The limit of quantification (LOQ) was 20.0-40.0 ng/mL and the limit of detection (LOD) was 3.0-25.0 ng/mL. With this method, low levels of ganoderic acids in the fruiting bodies of Ganoderma sinense and Ganoderma applanatum were accurately quantified for the first time. Importantly, the method allows unequivocal quantification of the five ganoderic acids in the spores and fruiting bodies of Ganoderma lucidum, whereas the previously published methods have lacked the capability. The method presented will be a powerful tool for quality control of Ganoderma lucidum and its related species.     

Rapid and quantitative determination of solanesol in Nicotiana tabacum by liquid chromatography-tandem mass spectrometry

A rapid and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method with multiple reaction monitoring (MRM) was developed for the determination of solanesol in Nicotiana tabacum. Sample preparation was performed by ultrasonic extraction with methanol for 20 min and then supernatant was extracted with hexane. The method used atmospheric pressure chemical ionization (APCI) detection in positive-ion mode. The separation of solanesol was performed on a Symmetry Shield RP18 column with a mixture of acetonitrile and isopropanol (1:1, v/v) containing 2mM ammonium acetate as mobile phase. Quantification of solanesol was performed by the standard addition method. The limit of quantification (LOQ) and limit of detection (LOD) of solanesol were, respectively, 5.0 ng/ml (S/N=10) and 1.5 ng/ml (S/N=3). The relative standard deviations of peak area were 0.89 and 1.12% for intra-day and inter-day, respectively. The recoveries of solanesol ranged from 97.72 to 99.67% and the corresponding R.S.D.s were less than 2.7%. Analysis took 5 min, making the method suitable for rapid determination of solanesol in N. tabacum. The proposed method has been successfully applied to the analysis of solanesol in various organs of N. tabacum.     

Determination of ionophore coccidiostats in feeding stuffs by liquid chromatography-tandem mass spectrometry. Part II. Application to cross-contamination levels and non-targeted feed

A fit to purpose multi-analyte method for the official control of six coccidiostats (monensin sodium, salinomycin sodium, narasin, lasalocid sodium, semduramicin sodium and maduramicin ammonium alpha) at cross-contamination concentration levels in poultry, cattle, pig and calf compound feed by liquid chromatography tandem mass spectrometry (LC-MS/MS) has been developed and in-house validated. The corresponding maximum levels have been recently introduced by European legislation. The method developed involved a simple extraction of the coccidiostats from the feed samples followed by centrifugation and filtration of the supernatants for all matrices. For calf feed an additional de-fattening step of the filtrated supernatants with n-hexane was necessary. The resulting supernatants were submitted to chromatographic analysis. The analytes were quantified by a modified approach of the standard additions technique applied to the extracts, hence allowing a workload comparable to matrix-matched standard calibration curves. A further simplification of this technique was reached by applying the same addition levels of the target analytes for different concentration ranging from 0.5× maximum level up to 2.5× maximum level (universal approach). The concentration independent intermediate precision expressed in terms of relative standard deviation varied between 3 and 12% (except for maduramicin ammonium alpha and semduramicin sodium up to 21%) and the recovery rates ranged from 80 to 111%, depending on the target analyte and matrix. The limits of detection (LOD) and limits of quantification (LOQ) were different for the various analyte/matrix/instrument combinations but all LOQs were in the 0.01-0.65 mg kg(-1) range, hence well below the target concentrations of each analyte. Based on the obtained method performance characteristics the method is considered fit for the intended purpose.     

Development of chiral liquid chromatography-tandem mass spectrometry isotope dilution methods for the determination of unconjugated and total S-equol in human plasma and urine

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods for the determination of unconjugated and total (conjugated plus unconjugated) S-equol in human plasma and urine were developed and validated. The separation of R and S enantiomers was achieved with a Chiracel OJ-H column operated in a normal phase mode using ethanol/hexane mobile phase components. Ionization of S-equol by negative ion electrospray generated the [M-H](-) ion whose response was augmented by post-column addition of ammonium hydroxide. A triple stage quadrupole mass spectrometer was used to measure the ion current generated from the dissociative transitions m/z 241→m/z 121 (S-equol) and m/z 245→m/z 123 (equol-d(4)). The determination of total S-equol included an additional deconjugation step involving incubation of the sample with sulfatase and glucuronidase. Average recovery for both unconjugated and total S-equol was 85% with no observable matrix effects. Linearity was established for unconjugated S-equol from 0.025ng/mL to 10ng/mL (plasma) and 0.20ng/mL to 200ng/mL (urine). The average coefficient of variation and accuracy per occasion was within ±15% of the theoretical concentration of S-equol. The method was used to measure the pharmacokinetics of S-equol in human plasma after an oral administration of a single 20mg dose of S-equol to three normal healthy volunteers.     

Simple and accurate quantitative analysis of ten antiepileptic drugs in human plasma by liquid chromatography/tandem mass spectrometry

A simple, accurate, and sensitive liquid chromatography (LC)-tandem mass spectrometry (MS/MS) method has been developed for the simultaneous quantification of 10 antiepileptic drugs (AEDs; gabapentin (GBP), levetiracetam (LEV), valproic acid (VPA), lamotrigine (LTG), carbamazepine-10,11-epoxide (CBZ-epoxide), zonisamide (ZNS), oxcarbazepine (OXC), topiramate (TPM), carbamazepine (CBZ), phenytoin (PHT)) in human plasma as a tool for drug monitoring. d₁₀-Phenytoin (d₁₀-PHT) and d_6-valproic acid (d₆-VPA) were used as internal standards for the positive- and negative-ionization modes, respectively. Plasma samples were precipitated by the addition of acetonitrile, and supernatants were analyzed on a C18 reverse-phase column using an isocratic elution. Detection was carried out in selected reaction monitoring (SRM) mode. The calibration curves were linear over a 50-fold concentration range, with correlation coefficients (r²) greater than 0.997 for all AEDs. The intra- and inter-day precision was less than 12%, and the accuracy was between 85.9 and 114.5%. This method was successfully used in the identification and quantitation of AEDs in patients undergoing mono- or polytherapy for epilepsy.     

超高效液相色谱串联质谱法快速测定尼扎替丁在人体血浆中的含量

目的: 建立超高效液相色谱串联质谱法测定人体血浆中尼扎替丁的含量。方法: 血浆以甲醇直接沉淀蛋白, 上清液用于目标物的检测;色谱柱为Waters ACQUITY UPLC BEH C₁₈;流动相为5 mmol·L^-1醋酸铵水溶液-甲醇 (65:35, V/V);流速:0.20 mL·min^-1;进样量:3 μL;柱温:35 ℃;样品室温度:6 ℃;采用电喷雾正离子模式电离, 尼扎替丁和内标雷尼替丁的监测离子分别为(m/z)332.2→155.0, (m/z)315.2→130.0。结果: 尼扎替丁的线性范围为10~3 000 ng·mL^-1, 平均回收率为93.2%~101.1%, 批内、批间RSD均小于15%。结论: 本研究建立的分析方法快速、准确、灵敏、简便, 适用于尼扎替丁人体药动学方面的研究。     

Structural elucidation of in vivo and in vitro metabolites of anisodine by liquid chromatography-tandem mass spectrometry

Liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESIMSn) was employed to investigate the in vivo and in vitro metabolism of anisodine. Feces, urine and plasma samples were collected after ingestion of 20 mg anisodine to healthy rats. Feces and urine samples were cleaned up by liquid-liquid extraction and solid-phase extraction procedures (C18 cartridges), respectively. Methanol was added to plasma samples to precipitate plasma proteins. Anisodine was incubated with homogenized liver and intestinal flora of rats in vitro, respectively, followed by extraction with ethyl acetate. LC-MSn was used for the separation and identification of the metabolites using C18 column with mobile phase of methanol/0.01% triethylamine solution (2 mM, adjusted to pH 3.5 with formic acid) (60:40, v/v). The results revealed that five metabolites (norscopine, scopine, alpha-hydroxytropic acid, noranisodine and hydroxyanisodine) and the parent drug existed in feces. Three new metabolites (dimethoxyanisodine, tetrahydroxyanisodine and trihydroxy-methoxyanisodine) were identified in urine. Four metabolites (norscopine, scopine, hydroxyanisodine and anisodine N-oxide) and the parent drug were detected in plasma. Two hydrolyzed metabolites (scopine and alpha-hydroxytropic acid) were found in rat intestinal flora incubation mixture, and two metabolites (aponoranisodine and anisodine N-oxide) were identified in homogenized liver incubation mixture.     

Development of a liquid chromatography/tandem mass spectrometry method for the quantitation of acetylcholine and related neurotransmitters in brain microdialysis samples

Monitoring concentrations of acetylcholine (ACh) in specific brain regions is important in understanding disease pathology, as well as in designing and evaluating novel disease-modifying treatments where cholinergic dysfunction is a hallmark feature. We have developed a sensitive and quantitative liquid chromatography/tandem mass spectrometry method to analyze the extracellular concentrations of ACh, choline (Ch) and (3-carboxylpropyl)-trimethylammonium (iso-ACh) in brain microdialysis samples of freely moving animals. One immediate advantage of this new method is the ability to monitor ACh in its free form without having to use a cholinesterase inhibitor in the perfusate. The separation of ACh, Ch, iso-ACh and related endogenous compounds was carried out based on cation exchange chromatography with a volatile elution buffer consisting of ammonium formate, ammonium acetate and acetonitrile. An unknown interference of ACh, which was observed in brain microdialysates from many studies, was well separated from ACh to ensure the accuracy of the measurement. Optimization of electrospray ionization conditions for these quaternary ammonium compounds achieved the limits of detection (S/N=3) of 0.2 fmol for ACh, 2 fmol for Ch and 0.6 fmol for iso-ACh using a benchtop tandem quadrupole mass spectrometer with moderate sensitivity. The limit of quantitation (S/N=10) was 1 fmol for ACh, 3 fmol for iso-ACh and 10 fmol for Ch. This method was selective, precise (<10% R.S.D.), and sensitive over a range of 0.05-10nM for ACh, 0.25-50 nM for iso-ACh and 15-3000 nM for Ch. To demonstrate that the developed method can be applied to monitoring changes in ACh concentrations in vivo, reference agents that have previously been shown to influence ACh levels were studied in rat dorsal hippocampus. This includes the 5-HT6 receptor antagonist, SB-271046, and the cholinesterase inhibitor, donepezil. Moreover, levels of ACh were demonstrated to be sensitive to infusion of tetrodotoxin (TTX) suggesting that the ACh being measured in vivo was of neuronal origin. Collectively, these biological data provided in vivo validation of this analytical method.     

Rapid and simultaneous measurement of midazolam, 1'-hydroxymidazolam and digoxin by liquid chromatography/tandem mass spectrometry: application to an in vivo study to simultaneously measure P-glycoprotein and cytochrome P450 3A activity

In order to simultaneously determine in vivo P-glycoprotein (P-gp) and Cytochrome P450 3A (CYP3A) activity, a new, rapid and sensitive liquid chromatography/tandem mass spectrometry (LC-MS/MS) method has been developed and fully validated to simultaneously determine midazolam (MDZ, as CYP3A substrate), 1'-hydroxymidazolam (1'-OHMDZ) and digoxin (DG, as P-gp substrate) in rat plasma using digitoxin as the internal standard (IS). After a single step liquid-liquid extraction with tert-butyl methyl ether/dichloromethane (75:25, v/v), analytes were subjected to LC-MS/MS analysis using positive electro-spray ionization (ESI(+)) under selected reaction monitoring mode (SRM). Chromatographic separation was performed on an XTerra MS C18 column (50mm×2.1mm, i.d. 3.5μm). The MS/MS detection was conducted by monitoring the fragmentation of 326.05 → 244.00 (m/z) for MDZ, 342.02 →168.01 (m/z) for 1'-OHMDZ, 798.33 → 651.36(m/z) for DG and 782.67 → 635.24 (m/z) for IS. The method had a chromatographic running time of 3min and linear calibration curves over the concentrations of 2-400ng/mL for MDZ and 1'-OHMDZ and 0.5-100ng/mL for DG. The recoveries of the method were 86.8-96.3% for MDZ, 84.6-86.4% for 1'-OH MDZ, and 81.7-85.1% for DG. The lower limit of quantification (LLOQ) of the method was 2ng/mL for MDZ and 1'-OHMDZ and 0.5ng/mL for DG. The intra- and inter-batch precision were less than 15% for all quality control samples at concentrations of 5, 50 and 320ng/mL for MDZ and 1'-OHMDZ and 1, 10 and 80ng/mL for DG. The validated LC-MS/MS method has been successfully used to analyze the concentrations of MDZ, 1'-OH MDZ and DG in rat plasma for simultaneous measurement of in vivo P-gp and CYP 3A activity.     

Effect of gender and cigarette smoking on urinary excretion of etheno DNA adducts in humans measured by isotope dilution gas chromatography/mass spectrometry

Endogenous formation of the promutagenic DNA adducts 1,N(6)-ethenoadenine (epsilon Ade) and 3,N(4)-ethenocytosine (epsilon Cyt) has been considered as biomarkers originated from lipid peroxidation. Elevated levels of epsilon Ade and epsilon Cyt were observed in cancer-prone tissues, suggesting the validity of these adducts in cancer risk assessment. The presence of DNA base adducts in biological fluids is considered to derive primarily from base excision repair (BER) systems. In this study, a modified gas chromatography/mass spectrometry (GC/MS) method is developed for simultaneous analysis of epsilon Ade and epsilon Cyt in human urine. After adjusting for creatinine concentration, urinary excretion of epsilon Ade, as well as epsilon Cyt, is much higher in 18 male smokers than in 10 male nonsmokers (p=0.003 for epsilon Ade and p=0.04 for epsilon Cyt). Furthermore, excretion of epsilon Ade and epsilon Cyt in 14 female nonsmokers is much higher than in 10 male nonsmokers (p=0.002 for epsilon Ade and p=0.005 for epsilon Cyt). These results suggest a statistically significant association between gender, as well as smoking, and excretion of epsilon Ade and epsilon Cyt. Moreover, urinary excretion of epsilon Ade in these 42 subjects correlates with that of epsilon Cyt (R(2)=0.6846, p<0.0001). Measurement of urinary epsilon Ade and epsilon Cyt excretion should provide valid noninvasive biomarkers for carcinogenesis and chemoprevention studies.     

胶体金法与液相色谱-质谱法MDMA检测比较

目的：通过胶体金法对药物滥用患者尿液中3,4-亚甲基二氧基甲基苯丙胺（MDMA）及代谢产物进行检测,并与液相色谱一质谱法检测结果进行比较以验证其检测效果。方法：选取广州市脑科医院物质依赖科收治的药物滥用患者,分别采用胶体金法和液相色谱一质谱法检测其尿液中MDMA及相关成分,计算分析胶体金法检测试剂的灵敏度、特异度、Youden指数与总符合率。结果：在以液相色谱-质谱法为金标准的检测结果对比中,胶体金法检测试剂的灵敏度100．00％,特异度98．88％,Youden指数0．9888,样本检测总符合率98．97％,Kappa=0．936,P=0．00。结论：与液相色谱-质谱法相比较,胶体金法具有操作简便、直观、快速、省时的特点,其特异性、敏感性较高,具有良好的检测效果。     

Determination of ionophore coccidiostats in feedingstuffs by liquid chromatography-tandem mass spectrometry Part I. Application to targeted feed

A new and fit to purpose multi-analyte method for the determination of six coccidiostats (monensin A, salinomycin, narasin composed of its principal component narasin A and its minor component narasin I, lasalocid, semduramicin and maduramicin) in poultry and cattle compound feed by liquid chromatography tandem mass spectrometry (LC-MS/MS) has been developed and in-house validated. The concentration level of the target analytes at which the validation experiments have been carried out varied between 1 and 9 mg kg(-1). The method developed involved a simple extraction of the coccidiostats from the feed samples followed by a clean-up by solid-phase extraction prior to chromatographic analysis. The analytes were quantified either by matrix-matched standards or by the standard addition technique, obtaining the following performance profile of the method for the various analyte/matrix combinations. When quantifying against matrix-matched standards, the concentration independent intermediate precision expressed in terms of relative percentage standard deviation varied between 4 and 10% and the relative percentage recovery rates ranged from 86 to 120%, depending on the target analyte and matrix. When using the standard addition technique, the corresponding values for the intermediate precision varied between 2 and 8% and the relative percentage recovery rate ranged from 73 to 115%. The limit of detection (LOD) and limit of quantification (LOQ) were different for the various analyte/matrix combinations but were in all cases below 0.014 and 0.046 mg kg(-1), respectively. Based on the obtained method performance characteristics, the method is considered suitable for the determination of ionophore coccidiostats in target feed. The main field of application of the validated method is to enforce European legislation regarding the authorisation of coccidiostats, focusing on the measurement at the authorised levels and at low level in feed during the withdrawal period at which the coccidiostats must not be added to the feed. Overall, the method proposed appears to be appropriate as a confirmatory method for the monitoring of these six ionophore coccidiostats and can therefore be considered as complementary to the official HPLC-UV methods.     

Simultaneous detection and quantification of 3-nitrotyrosine and 3-bromotyrosine in human urine by stable isotope dilution liquid chromatography tandem mass spectrometry

Nitration and bromination of proteins, giving rise to the respective 3-nitrotyrosine (3NT) and 3-bromotyrosine (3BT), are implicated in asthma, allergic inflammatory disorders, and cancer. We have developed an isotope dilution liquid chromatography electrospray ionization tandem mass spectrometry (LC/MS/MS) assay for simultaneous analysis of protein-bound 3NT and 3BT in human urine. The detection limits (S/N = 3) were 10 pg (44 fmol) for 3NT and 5.0 pg (19 fmol) for 3BT injected on-column. The average levels of protein-bound 3NT and 3BT in 23 healthy individuals were 9.7 ± 11.0 (mean ± S.D.) in 10⁵ tyrosine and 4.4 ± 3.9 (mean ± S.D.) in 10³ tyrosine, respectively, using this highly sensitive LC/MS/MS under the selective reaction monitoring mode. Furthermore, the levels of urinary 3NT and 3BT show a statistically significant correlation (R² = 0.55, p = 0.0065, n = 23). The high specificity and accuracy of this LC/MS/MS method render it a valuable tool in measurement of 3NT and 3BrT in the human urinary protein as promising noninvasive biomarkers for protein tyrosine nitration and bromination in vivo.     

High-throughput determination of mono- and di(2-ethylhexyl)phthalate migration from PVC tubing to drugs using liquid chromatography-tandem mass spectrometry

The risk assessment of di(2-ethylhexyl) phthalate (DEHP) that migrated from polyvinyl chloride (PVC) medical devices is an important issue for hospitalized patients. Many studies have been conducted to determine the level of DEHP migration. A recent report has indicated that DEHP in blood bags was hydrolyzed by esterase to mono(2-ethylhexyl) phthalate (MEHP). Therefore, a method for the simultaneous determination of DEHP and MEHP was developed. The migration of DEHP and MEHP from PVC tubing to drugs was examined. Although we detected MEHP in the drugs, we found no enzymatic activity involved in the migration process. Some reports have indicated that hydrolysis may have occurred during sterilization by autoclaving. However, we did not perform any heat treatment. It is speculated that the MEHP migrated directly from the PVC tubing. The simultaneous determination of DEHP and MEHP is required for risk assessment, as MEHP may be even more toxic than the parent compound.     

Liquid chromatography/tandem mass spectrometry utilizing ion-molecule reactions and collision-activated dissociation for the identification of N-oxide drug metabolites

A liquid chromatography/tandem mass spectrometry (LC/MS³) method based on ion-molecule reactions and collision-activated dissociation (CAD) is presented for the identification of analytes with the N-oxide functional group directly in mixtures. Tri(dimethylamino)borane (TDMAB) rapidly and selectively derivatizes protonated N-oxides in a modified commercial linear quadrupole ion trap (LQIT) mass spectrometer to yield a distinct product ion (adduct—(CH₃)₂NH). The LQIT was outfitted with an external reagent-mixing manifold that allows TDMAB to be mixed with the helium buffer gas used in the trap. The derivatized analytes are readily identified on the basis of a shift of 98 Th (Thomson) relative to the m/z value of the protonated analyte. Further probing of the derivatized analytes via isolation followed by CAD can be used to confirm the presence of an N-oxide, and distinguish between aliphatic and aromatic tertiary N-oxides. Since the ion-molecule reaction is fast, these experiments can be accomplished on the same time scale as typical CAD-based MSⁿ experiments, thus maintaining the duty cycle of the instrument for this type of experiment. To demonstrate real world applicability, the method was tested on real active pharmaceutical ingredients and their derivatives.     

The simultaneous determination of coumarins in Angelica gigas root by high performance liquid chromatography-diode array detector coupled with electrospray ionization/mass spectrometry

An high performance liquid chromatography-diode array detector coupled with electrospray ionization/mass spectrometry (HPLC-DAD/MS) based method has been developed for the simultaneous determination of nine coumarin compounds, nodakenin (1), peucedanone (2), marmesin (3), decursinol (4), 7-hydroxy-6-(2R-hydroxy-3-methylbut-3-enyl)coumarin (5), demethylsuberosin (6), decursin (7), decursinol angelate (8) and isoimperatorin (9) in the Korean medicinal herb, Cham-Dang-Gui, the dried root of Angelica gigas (Umbelliferae). The methanol extracts were analyzed by HPLC using a reversed-phase C18 column (5 microm, 4.5 mm x 250 mm) using a gradient acetonitrile-water solvent system at a flow rate of 1.0 ml/min. The analysis of six coumarins (1, 3, 4 and 6-8) with DAD was done at 330 nm and showed excellent linearity (r(2)=0.998-0.999) in a range of 0.2-250 microg/ml for all the compounds. The average recoveries (n=3) were between 96.5% and 110.8%. Identification of each peak was also discussed with the electrospray ionization multi-stage tandem mass spectroscopy (ESI-MS(n)). The amount of these coumarin compounds was evaluated in A. gigas samples. Meanwhile, three coumarins (2, 5 and 9) could not been quantified by DAD because these peaks were overlapped with others. Determination of these compounds could be successfully accomplished with the HPLC-ESI/MS in selected ion monitoring/selected reaction monitoring mode.     

Use of liquid chromatography-mass spectrometry and a chemical cleavage reaction for the structure elucidation of a new sildenafil analogue detected as an adulterant in an herbal dietary supplement

An herbal dietary supplement, marketed as a natural product for the enhancement of sexual function, was analyzed by HPLC with photodiode array and mass spectral detection and found to contain a compound related to the synthetic phosphodiesterase-5 (PDE-5) inhibitors. Based on UV spectra, mass spectra and direct infusion MS(n), the structure of the compound was tentatively identified as a sildenafil analogue in which the sulfonyl group had been replaced with an acetyl group. This new analogue is similar to acetildenafil, a previously reported sildenafil analogue, but differs in that it contains an N-methyl group where acetildenafil contains an N-ethyl group. The structure of the unknown was unequivocally established by chemical cleavage of the phenacylamine group of the molecule to generate N-methylpiperazine; other cleavage products matched those generated from acetildenafil. Since the new compound has one less CH(2) group than acetildenafil, it was named nor-acetildenafil.