Ho Jun Kim

Simple and accurate quantitative analysis of seven prohibited threshold substances in human urine by liquid chromatography/tandem mass spectrometry in doping control.

A simple and accurate liquid chromatography/tandem mass spectrometry (LC/MS/MS) method has been developed and validated for the quantitative determination of ephedrine, pseudoephedrine, methylephedrine, cathine, salbutamol, morphine and epitestosterone in human urine. Urine samples were spiked with internal standard and diluted with acetonitrile. After centrifugation, the supernatants were directly analyzed by LC/MS/MS using the selected reaction monitoring (SRM) mode. The linearity, intra- and inter-day precision, accuracy, limit of detection (LOD) and limit of quantification (LOQ) were evaluated and the method was found to be accurate and reproducible for the quantitation of threshold substances. When the method was applied to the analysis of blind urine samples for the proficiency test, the results were close to the nominal concentrations, within 87.7-106.6% of nominal values, suggesting that the developed methods can be successfully applied to routine doping analyses.

Simultaneous analysis of 210 prohibited substances in human urine by ultrafast liquid chromatography/tandem mass spectrometry in doping control

RATIONALE Doping analysis is a two-step process consisting of a screening step for prohibited substances and a confirmation step to verify the presence of specific substances found during the screening. The entire process must be performed within a limited time period, but traditional screening procedures commonly employ separate analytical methods for each class of prohibited substances being screened and thus require a great deal of human resources and instrumentation. A single simple and rapid multiresidue analytical method that could accommodate multiple classes of prohibited substances would be extraordinarily useful in doping analyses. METHODS Urine samples were extracted via two consecutive liquid-liquid extractions at different pH values following enzymatic hydrolysis. Analyses were performed by ultrafast liquid chromatography/triple-quadrupole mass spectrometry with polarity switching and time-dependent selected reaction monitoring. RESULTS We developed a rapid multiresidue screening and confirmation method for efficient high-throughput doping analyses. The present method was validated with regard to the limits of detection (0.01-100.0 ng/mL for screening analyses and 0.2-500.0 ng/mL for confirmation assays), matrix effects (48.9-118.9%), recovery (20.6-119.7%) and intra- (0.6-17.6%) and inter-day (4.0-20.0%) precision. CONCLUSIONS A multiresidue analytical method was developed and validated for screening and confirming the presence of performance-enhancing drugs. A total of 210 substances from diverse classes of prohibited substances were successfully identified with an analytical run time of 10 min.

Simultaneous ionization and analysis of 84 anabolic androgenic steroids in human urine using liquid chromatography‐silver ion coordination ionspray/triple‐quadrupole mass spectrometry

Metal ion coordination ionspray (M(+) CIS) ionization is a powerful technique to enhance ionization efficiency and sensitivity. In this study, we developed and validated an analytical method for simultaneous ionization and analysis of 84 anabolic androgenic steroids (65 exogenous and 19 endogenous) using liquid chromatography-silver ion coordination ionspray/triple-quadrupole mass spectrometry (LC-Ag(+) CIS/MS/MS). The concentrations of silver ions and organic solvents have been optimized to increase the amount of silver ion coordinated complexes. A combination of 25 μM of silver ions and methanol showed the best sensitivity. The validation results showed the intra- (0.8-9.2%) and inter-day (2.5-14.9%) precisions, limits of detection (0.0005-5.0 ng/mL), and matrix effect (71.8-100.3%) for the screening analysis. No significant ion suppression was observed. In addition, this method was successfully applied to analysis of positive samples from suspected abusers and useful for the detection of the trace levels of anabolic steroids in human urine samples.

Simple quantitation of formoterol and 11-nor-Δ9-tetrahydrocannabinol-9-carboxylic acid in human urine by liquid chromatography-tandem mass spectrometry in doping control

11-nor-Δ(9)-tetrahydrocannabinol-9-carboxylic acid (THC-COOH) and formoterol are newly revised prohibited threshold substances (150 ng/mL for THC-COOH and 40 ng/mL for formoterol) by the World Anti-Doping Agency (WADA). In continuation of our direct quantitation work of the prohibited threshold substances, direct LC-MS/MS methods combined with a simple sample preparation procedure have been developed and validated for the measurement of these two threshold substances in urine samples. After the enzymatic hydrolysis of urine samples, the resulting samples were diluted with acetonitrile and centrifuged. The supernatant was directly analyzed by LC-MS/MS using the selected reaction monitoring mode. The calibration curve range of the assay was ranged over 50-200% of the threshold value according to WADA guidelines. The limit of detection and limit of quantification were 6.1 and 18.4 ng/mL for THC-COOH and 2.0 and 6.2 ng/mL for formoterol, respectively. Intra- and inter-day precisions were between 2.08% and 7.28% and the accuracies ranged from 95.16% to 104.49%. The present methods were successfully applied to the analysis of the proficiency test samples.

Sensitivity of GC-EI/MS, GC-EI/MS/MS, LC-ESI/MS/MS, LC-Ag(+) CIS/MS/MS, and GC-ESI/MS/MS for analysis of anabolic steroids in doping control.

This study compared the sensitivity of various separation and ionization methods, including gas chromatography with an electron ionization source (GC-EI), liquid chromatography with an electrospray ionization source (LC-ESI), and liquid chromatography with a silver ion coordination ion spray source (LC-Ag(+) CIS), coupled to a mass spectrometer (MS) for steroid analysis. Chromatographic conditions, mass spectrometric transitions, and ion source parameters were optimized. The majority of steroids in GC-EI/MS/MS and LC-Ag(+) CIS/MS/MS analysis showed higher sensitivities than those obtained with other analytical methods. The limits of detection (LODs) of 65 steroids by GC-EI/MS/MS, 68 steroids by LC-Ag(+) CIS/MS/MS, 56 steroids by GC-EI/MS, 54 steroids by LC-ESI/MS/MS, and 27 steroids by GC-ESI/MS/MS were below cut-off value of 2.0 ng/mL. LODs of steroids that formed protonated ions in LC-ESI/MS/MS analysis were all lower than the cut-off value. Several steroids such as unconjugated C3-hydroxyl with C17-hydroxyl structure showed higher sensitivities in GC-EI/MS/MS analysis relative to those obtained using the LC-based methods. The steroids containing 4, 9, 11-triene structures showed relatively poor sensitivities in GC-EI/MS and GC-ESI/MS/MS analysis. The results of this study provide information that may be useful for selecting suitable analytical methods for confirmatory analysis of steroids.

Simultaneous Determination of Synthetic Phosphodiesterase-5 Inhibitors in Dietary Supplements by Liquid Chromatography-High Resolution/Mass Spectrometry

After success of sildenafil for the treatment of erectile dysfunction, a large number of its analogues have been approved from FDA. Recently, the illegal dietary supplements which include sildenafil, vardenafil, tadalafil, or analogues of these drugs as ingredient have been widely distributed. Therefore, the determination of the residue of synthetic phosphodi- esterase-5 (PDE-5) inhibitors in dietary supplements is highly required due to indiscriminate and unintentional overdose caused nausea, chest pains, fainting and irregular heartbeat. In this paper, we report a rapid and sensitive analytical method for the simultaneous determination of nine phosphodiesterase-5 inhibitors by liquid chromatography-high resolution mass spectrome- try. The present method was found to be accurate and reproducible with 40 µg/g of the limit of quantification for the nine PDE-5 inhibitors. The developed method can be successfully applied to the analysis of the seven illegal dietary supplements.

Relationships between structure, ionization profile and sensitivity of exogenous anabolic steroids under electrospray ionization and analysis in human urine using liquid chromatography–tandem mass spectrometry

The relationships between the ionization profile, sensitivity, and structures of 64 exogenous anabolic steroids (groups I-IV) was investigated under electrospray ionization (ESI) conditions. The target analytes were ionized as [M + H](+) or [M + H-nH2 O](+) in the positive mode, and these ions were used as precursor ions for selected reaction monitoring analysis. The collision energy and Q3 ions were optimized based on the sensitivity and selectivity. The limits of detection (LODs) were 0.05-20 ng/mL for the 64 steroids. The LODs for 38 compounds, 14 compounds and 12 compounds were in the range of 0.05-1, 2-5 and 10-20 ng/mL, respectively. Steroids including the conjugated keto-functional group at C3 showed good proton affinity and stability, and generated the [M + H](+) ion as the most abundant precursor ion. In addition, the LODs of steroids using the [M + H](+) ion as the precursor ion were mostly distributed at low concentrations. In contrast, steroids containing conjugated/unconjugated hydroxyl functional groups at C3 generated [M + H - H2 O](+) or [M + H - 2H2 O](+) ions, and these steroids showed relatively high LODs owing to poor stability and multiple ion formation. An LC-MS/MS method based on the present ionization profile was developed and validated for the determination of 78 steroids (groups I-V) in human urine.

Simultaneous qualitative and quantitative method using liquid chromatography selected reaction monitoring-triggered quantitation-enhanced data-dependent tandem mass spectrometry for the identification and classification of amphetamine-type stimulant abusers in human urine.

Amphetamine (AP) and amphetamine-type stimulants, methamphetamine (MA) and N,N-dimethylamphetamine (DMA), are known as central nervous system stimulants, and their abuse throughout the world has recently increased. Since it is difficult to physically distinguish among AP, MA and DMA, analysts may not be aware of what abusers have administered. In this study, following the detection of specific metabolites of AP, MA and DMA as biomarkers in abuser urines, a rapid and sensitive method was developed for the identification and classification of AP-type stimulants abusers. After the simple filtration of the urine samples, the samples were directly analyzed using a liquid chromatography/tandem mass spectrometry system with selected reaction monitoring (SRM)-triggered quantitation-enhanced data-dependent MS/MS (QED-MS/MS) for the simultaneous qualitative and quantitative analysis of p-hydroxy AP, p-hydroxy MA, p-hydroxy DMA, AP, MA, DMA and DMA N-oxide. The determination of p-hydroxy AP, p-hydroxy MA, AP, MA, DMA and DMA N-oxide was accurate and reproducible, with the limits of quantitation of 5 ng/mL in urine. When applied to the urine samples of suspected AP-type stimulants abusers, the abused drugs were precisely identified between MA and DMA abusers.

LC-MS/MS Method for Simultaneous Analysis of Growth Hormone-Releasing Peptides and Secretagogues in Human Urine

Growth hormone (GH)-releasing peptides (GHRPs) and GH secretagogues (GHSs) are listed in the World Anti-Doping Agency (WADA) Prohibited List. In the present study, we developed and validated a method for the simultaneous analysis of seven GHRPs (alexamorelin, GHRP-1, -2, -4, -5, -6, and hexarelin) and three GHSs (anamorelin, ibutamoren, and ipamorelin) in human urine. Method validation was performed at minimum required performance levels specified by WADA technical documents (2 ng/mL) for all substances, and the method was validated with regard to selectivity (no interference), linearity (R2 > 0.9986), matrix effects (50.0%-141.2%), recovery (10.4%-100.8%), and intra- (2.8%-16.5%) and inter-day (7.0%-22.6%) precisions. The limits of detection for screening and confirmation were 0.05-0.5 ng/mL and 0.05-1 ng/mL, respectively.

Ultra-fast Generic LC-MS/MS Method for High-Throughput Quantification in Drug Discovery

An ultra-fast generic LC-MS/MS method was developed for high-throughput quantification of discovery pharmacok- inetic (PK) samples and its reliability was verified. The method involves a simple protein precipitation for sample preparation and the analysis by ultra-fast generic LC-MS/MS with the ballistic gradient program and selected reaction monitoring (SRM) mode. Approximately 290 new chemical entities (NCEs) (over 10,000 samples) from 5 therapeutic programs were analyzed. The calibration curves showed good linearity in the concentration range of 1, 2 or 5 to 2000 ng/mL. No significant ion suppres- sion was observed in the elution region of all the NCEs. When approximately 300 plasma samples were continuously analyzed, the peak area of internal standard was constant and reproducible. In the repeated analysis of samples, the plasma concentrations and the area under the curve (AUC) were consistent with the results from the first analysis. These results showed that the present ultra-fast generic LC-MS/MS method is reliable in terms of selectivity, sensitivity, and reproducibility and could be useful for high-throughput quantification and other bioanalysis in drug discovery.