Simultaneous determination of 11 nootropic substances potentially adulterated in dietary supplements for improving brain function using ultra-performance liquid chromatography and liquid chromatography-quadrupole-time-of-flight mass spectrometry.

Recently, the demand for improved brain function and concentration has increased in the dietary supplement market. However, to artificially enhance their pharmacological efficacy, dietary supplements may be illegally adulterated with unauthorised substances. Therefore, we developed a rapid and accurate method to simultaneously determine 11 nootropic substances using an ultra-high-performance liquid chromatography (UPLC) system equipped with a photodiode array (PDA) detector. In addition, sample preparation procedures were semi-optimised for various types of matrices, including solid (hard capsule, tablet, powder, and pill) and liquid (oil and extract) samples. The method was validated to determine the limit of detection (LOD), limit of quantification (LOQ), method detection limit (MDL), method quantitation limit (MQL), specificity, linearity, precision, accuracy, recovery, stability, and matrix effects. The validation results satisfied international validation guideline requirements. To test the applicability of the method, 55 real samples advertised as effective brain health, memory, and cognition supplements were analysed. Among the real samples, vinpocetine (2.483 and 7.296 µg/g), and kavain (69-44.056 µg/g) were detected. In addition, the detected compounds were confirmed by comparing their fragmentation patterns with those of the reference standards using liquid chromatography-quadrupole-time-of-flight mass spectrometry (LC-QTOF/MS). In conclusion, the UPLC-PDA method not only rapidly and accurately quantifies illegal nootropics but also enables the pre-emptive investigation and identification of 11 nootropic substances in illegal dietary supplements to protect public health.

Intercomparison study of fragmentation pathways and fragment ion structures for screening of illegal drugs and their novel analogues used to adulterate dietary supplements using liquid chromatography/quadrupole time-of-flight mass spectrometry.

RATIONALE: As public interest in health and immunity has increased in recent years, so has the demand for dietary supplements. However, supplements adulterated with illegal drugs and their novel analogues are being sold even as the pharmacological efficacies of these drugs are being advertised. Since the use of these illegal compounds can have serious side effects, they pose a risk to public health. Hence, in this study, we propose a strategy for proactively testing drugs and novel analogues that may be added to dietary supplements illegally. METHODS: The optimal conditions for liquid chromatography/quadrupole time-of-flight mass spectrometry were explored to determine the fragmentation patterns for 60 compounds. The optimal conditions were established by comparing the areas and heights of the precursor ion peaks at a fragmentor voltage of 125 or 175 V. Furthermore, the optimized spectra were acquired using collision energies of 1 to 50 eV. The energy value was selected based on the condition that the mass error of the precursor ions is 10 ppm or lower. RESULTS: The fragmentation pathway of each product ion and its chemical structure were predicted and determined. In addition, the obtained structural information was used to screen 18 seized samples. Based on the precursor ions and the corresponding fragmentation patterns, the unknown compounds present in the samples were identified as desulfonylchlorosildenafil and propoxyphenylthiohydroxy homosildenafil. CONCLUSIONS: We obtained mass spectrometry-based information for various compounds by predicting the fragmentation pathways and chemical structures of their fragment ions. Subsequently, based on the obtained structural information, we tested several seized samples and were able to detect two novel analogues in four of the samples. Therefore, the proposed approach is suitable for quickly and accurately identifying the unknown compounds detected in real-world samples.

Application of predicted fragmentation pathways and fragment ion structures for detecting steroids and selective androgen receptor modulators in dietary supplements using liquid chromatography-quadrupole time-of-flight mass spectrometry.

RATIONALE: Dietary supplements advertised to strengthen muscles have earned fame among athletes. However, several products containing unauthorized compounds are often detected, which can cause a public health risk. Particularly, steroids and selective androgen receptor modulators (SARMs) can cause serious side effects as hormone modulators. In this study, we analyzed 15 steroids and 20 SARMs using liquid chromatography-quadrupole time-of-flight mass spectrometry (LC/QTOFMS) to provide fundamental information about fragmentation pathways and fragment ion structures. METHODS: The optimal conditions of LC/QTOFMS were explored to obtain fragmentation patterns for each compound. The optimal conditions were established by comparing the area and height of the precursor ion peak at 125 or 175 V as a fragmentor energy. Furthermore, the optimized spectra were acquired by applying collision energy ranging from 1 to 50 eV. The energy value was selected under the condition that the mass error of precursor ions was less than 10 ppm. RESULTS: The 35 compounds were classified on the basis of their chemical core structures: arylpropionamide (3 compounds), quinolinone (2), pyrrolidinylbenzonitrile (1), indole (2), tropanol (2), phenylaxadaizole (1), hydantoin (2), phenylthiazole (1), nitrothiophene (1) and steroidal derivative (20). Fragmentation pathways and the chemical structure of each product ion were predicted and identified. Furthermore, the obtained structural information was applied to screen seized samples. As a result, 10 seized samples were confirmed to contain one or more SARMs by comparing each precursor ion and fragmentation pattern. CONCLUSIONS: The application to real samples for accurate screening indicated that the same fragmentation patterns and product ions as one or more SARM standards were detected and identified in the seized samples advertised as muscle building. Therefore, this study can contribute to ensuring the safety of public health through providing fundamental information about the risk of illegal adulteration.

Simultaneous screening of 21 potential adulterants in dietary supplements for the treatment of prostate diseases using ultra-performance liquid chromatography and liquid chromatography-electrospray ionization-tandem mass spectrometry.

Reports of the number of adulteration cases using illegal therapeutic substances in dietary supplements have increased. In recent years, various dietary supplements are being distributed that exaggerate the efficacy of treatment for prostate-related diseases. To develop the preemptive monitoring method, we selected 21 prostate-related therapeutic substances and optimized the simultaneous ultra-performance liquid chromatography and liquid chromatography-electrospray ionization-tandem mass spectrometry and pretreatment procedures for various types of matrices including solid, liquid, and soft capsule samples. The methods were validated by determining the specificity, linearity, limit of detection, limit of quantification, method detection limit, method quantitation limit, precision, accuracy, recovery, stability, and matrix effect. The simultaneous methods were validated according to the international guidelines. In addition, using the validated methods, 81 real samples, which were searched and purchased by focusing on promotional phrases, such as prostate and prostatic hyperplasia, were successfully screened. As a result, sildenafil and tadalafil were detected in one seized capsule sample (5.15 and 14.6 mg/g, respectively). Synthetically, our approach could be useful for the determination of illegal therapeutic substances potentially adulterated in various types of dietary supplements.

Application of a simultaneous screening method for the detection of new psychoactive substances in various matrix samples using liquid chromatography/electrospray ionization tandem mass spectrometry and liquid chromatography/quadrupole time-of-flight mass spectrometry.

RATIONALE: Recently, new psychoactive substances (NPS) have emerged as a public health risk. Particularly, their chemical structures are modified to avoid detection. Synthetic NPS with effects similar to those of illegal drugs have been recently detected and synthesized worldwide, including MDMB-FUBINACA and APINAC, making it essential to rapidly and accurately detect NPS. METHODS: Fourteen NPS with similar structures were selected and their structures identified using 1 H and 13 C NMR spectroscopy. Additionally, we proposed the fragmentation pattern of each compound using liquid chromatography/quadrupole time-of-flight mass spectrometry (LC/QTOF-MS). A simultaneous analytical method using liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) was also developed and applied to real samples to detect the 14 NPS. The method was validated based on the specificity, linearity, limit of detection (LOD), limit of quantification (LOQ), precision, accuracy, matrix effect, and stability according to international validation guidelines. RESULTS: The established method was used to screen 65 different matrix samples using LC/ESI-MS/MS. By comparing the calculated product ion ratios with those of standards, 2C-B in one of the real samples and 5F-MDMB-PICA in 20 samples were identified. For re-confirmation of detected compounds, the fragmentation pattern of each compound was compared with that of each standard using LC/QTOF-MS. CONCLUSIONS: In this study, LC/QTOF-MS data were used to elucidate the structures and fragmentation patterns of 14 NPS. A simultaneous method was developed using LC/ESI-MS/MS, which was applied to 65 real samples. The presented method and results can assist in ensuring the safety of public health from illegal adulteration.

Simultaneous screening of dietary supplements for 25 anti-hyperlipidemic substances using ultra-performance liquid chromatography and liquid chromatography/electrospray ionization tandem mass spectrometry.

RATIONALE: Recently, illegal dietary supplements containing unauthorized compounds have been seized and internationally publicized with a warning to avoid their consumption. This adulteration can be a serious threat to public health because of insufficient and reliable safety data as well as their undesirable side effects. It is, therefore, essential to rapidly and accurately develop and simultaneously validate analytical methods for these unauthorized anti-hyperlipidemic substances. METHODS: Dietary supplements were screened simultaneously for 25 anti-hyperlipidemic drugs using an ultra-high-performance liquid chromatography (UPLC) system with a photodiode array (PDA) detector and LC/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS). The method validation, according to ICH guidelines, considered specificity, linearity, limit of detection (LOD), limit of quantification (LOQ), precision, accuracy, matrix effects, and stability for solid and liquid blank samples. The established UPLC-PDA and LC/ESI-MS/MS methods were applied to screen 103 dietary supplements for 25 anti-hyperlipidemic substances. RESULTS: Using the validated methods, the screened samples were found to contain peaks with similar retention times and PDA spectra. By comparing the calculated precursor-product ion ratios with those of standards, lovastatin and lovastatin acid were detected at concentrations from LOQ to 4.12 mg/g and LOQ to 9.65 mg/g, respectively. CONCLUSIONS: The developed UPLC-PDA and LC/ESI-MS/MS analytical methods were applied to 103 real samples, of which 13 samples were found to contain lovastatin and lovastatin acid. In view of the increasing demand for dietary supplements in the treatment of hyperlipidemic diseases, widespread use of these methods will contribute to consumer health by ensuring the safety of dietary supplements.

New lignan tyramide, phenolics, megastigmanes, and their glucosides from aerial parts of New Zealand spinach, Tetragonia tetragonoides.

Twenty compounds, including a new lignan amide, were isolated from the aerial parts of New Zealand spinach, Tetragonia tetragonoides (Pall.) Kuntze, which is an edible halophyte. These compounds were identified by mass spectrometry and nuclear magnetic resonance experiments to be N-2,3-dihydroxy-3-(3,4-dihydroxyphenol)tyramine (new compound), methyl 4-hydroxybenzoate, syringaldehyde, ethyl 4-hydroxybenzoate, 3,4-dihydroxybenzoic acid, 2,3-dihydroxybenzoic acid, coniferyl alcohol, methyl caffeoate, trans- and cis-coumaroyl-ß-d-glucopyranosides, trans- and cis-feruloyl-ß-d-glucopyranosides, caffeic acid, staphylionoside E, canabiside D, apocyanol A, megastima-5,7-diene-3,4,9-triol, 1-O-oleoyl-3-O-ß-d-galactopyranosyl-sn-glycerol, 5,5'-dimethyl-lariciresinol, and kaempferol 3-O-ß-d-glucopyranoside. These compounds were identified in New Zealand spinach for the first time.

Simultaneous separation and determination of 20 potential adulterant antigout and antiosteoporosis pharmaceutical compounds in herbal food products using LC with electrospray ionization MS/MS and LC with quadrupole-time-of-flight MS.

An analytical method for the simultaneous and reliable determination of 20 antigout and antiosteoporosis pharmaceutical compounds in adulterated health food products was developed using liquid chromatography with electrospray ionization tandem mass spectrometry and liquid chromatography with quadrupole-time-of-flight mass spectrometry. The method was validated through the determination of specificity, linearity, limit of detection, and limit of quantification, method detection limit, method quantitation limit, precision, accuracy, recovery, and stability. The matrix effect was also determined. The validation results of the developed method are as follows: for solid and liquid blank samples, limits of detection ranged from 0.05 to 5.00 ng/mL and limits of quantification ranged from 0.15 to 15.00 ng/mL. Linearity was acceptable, and the correlation coefficients (R2 ) were ≥0.99 for all target compounds. Both intra and interday precision were less than 9.16% RSD, and accuracies ranged from 95.31 to 116.68%. Mean recoveries for different types of dietary supplements classified as powders, liquids, tablets, and capsules were found to be 80.81 to 117.62% with less than 15.00% relative standard deviation. The stability of the standard mixture solution was less than 11.72% relative standard deviation after 48 h. By the proposed method, the presence of dexamethasone was determined in seized herbal food products at concentrations that ranged from 126 to 215 µg/g.

Phenolics, acyl galactopyranosyl glycerol, and lignan amides from Tetragonia tetragonioides (Pall.) Kuntze.

Eleven antioxidative compounds, including five lignin amides, were isolated from the aerial part of Tetragonia tetragonioides (New Zealand spinach) using 1,1-diphenyl-2-picrylhydrazyl radicalscavenging assay-guided purification. The structures were determined by nuclear magnetic resonance and electrospray ionization-mass spectroscopy. These compounds were identified as methyl linoleate (1), methyl coumarate (2), methyl ferulate (3), 1-O-stearoyl-3-O-ß-D-galactopyranosyl-sn-glycerol (4), 1-O-caffeoyl-ß-D-glucopyranoside (5), N-trans-caffeoyltyramine (6), cannabisin B (7), cannabisin A (8), Ntrans-feruloyltyramine (9), N-cis-feruloyltyramine (10), and N-trans-sinapoyltyramine (11). Compounds 1, 2, 4, 5, and 8-11 were isolated for the first time from this plant.