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. 2020 Sep 24;25(19):4399. doi: 10.3390/molecules25194399

Multi-Class Determination of 64 Illicit Compounds in Dietary Supplements Using Liquid Chromatography–Tandem Mass Spectrometry

Dasom Shin 1, Hui-Seung Kang 1,*, Hyungsoo Kim 1, Guiim Moon 1
PMCID: PMC7583945  PMID: 32987895

Abstract

In this work, liquid chromatography–tandem mass spectrometry (LC-MS/MS) method was developed and validated for screening and confirmation of 64 illicit compounds in dietary supplements. The target compounds were illegally used pharmaceutical drugs, prohibited compounds, and not authorized ingredients for different therapeutics (sexual enhancement, weight loss, muscular strengthening, and relaxing products). The validation procedure was performed to evaluate selectivity, linearity, limit of detection (LOD), limit of quantification (LOQ), accuracy, and precision according to the Association of Official Analytical Chemists guidelines. The linearity was >0.98 in the range of 0.5–200 µg L−1. The LOQs were in the range 1–10 µg kg−1 for all target compounds. The accuracy (expressed as recovery) was 78.5–114%. The precision (expressed as the relative standard deviation) was below 9.15%. The developed method was applied for the determination of illicit compounds in dietary supplements collected from websites. As a result, the total detection rate was 13.5% (27 samples detected in 200 samples). The concentrations of detected samples ranged from 0.51 to 226 mg g−1. The proposed methodology is suitable for monitoring the adulteration of illicit compounds in dietary supplements.

Keywords: multi-class, illegal compounds, overseas direct purchase, analytical method, LC-MS/MS

1. Introduction

Consumer demand for dietary supplements that support healthy lifestyles has steadily increased worldwide. In the last decade, illegal adulterants of pharmaceuticals, unapproved drugs, and prohibited ingredients have increased and posed a global challenge in safety control of dietary supplements [1]. The easy access of Internet, overseas direct purchase, along with an increasingly complex global supply chain, has led to the widespread illegal drug distribution worldwide [2]. The abuses of illicit drugs cause adverse and side effects, such as heart attack, psychosis, hypertension, hallucination, tachycardia, agitation, vomiting, seizures, and convulsions [3]. The adulterated illicit compounds in dietary supplements have serious adverse health effect to consumers owing to misuse, overuse, or interaction with other medications [4]. Thus, continuous monitoring studies is the first priority to protect consumer health and safety management of dietary supplements [5].

HPLC equipped with ESI-MS/MS is a well-established and widespread technique for the analysis of various compounds illegally used to adulterants of sexual enhancement and weight loss compounds in dietary supplements [6,7,8]. Due to the increase in the adulteration of illegal compounds in dietary supplements, several analytical methods have been previously reported using liquid chromatography tandem mass spectrometry (LC-MS/MS) [9,10,11,12]. In addition, the multi-class analytical method using LC–MS/MS is widely applied for reliable quantitation and confirmation of illicit compounds [13,14].

In this work, we developed and validated the multi-class determination of 64 illicit compounds in dietary supplements using LC-MS/MS referred to previous studies. The target compounds were a variety of potential chemicals that could be illegally added into dietary supplements including pharmaceutical drugs, prohibited compounds, and not authorized ingredients according to Korean food legislation (Table S1). To the best of our knowledge, this is the first study to use an analytical method for the simultaneous determination of 64 illicit compounds and to control the custom clearance of illegal substances in Korea. The developed method was applied to real samples of dietary supplements collected by overseas direct purchase. The proposed multi-class methods for the screening and confirmation of compounds have been used for routine monitoring in dietary supplements.

2. Results and Discussion

2.1. Optimization of LC-MS/MS Conditions

The chromatographic separation was optimized by thoroughly adjusting the LC parameters such as symmetric peaks, column temperature, mobile phase, and flow rate, based on a previous study [5,15,16]. The optimal separation of target compounds was achieved using 0.1% formic acid in aqueous mobile phase and 0.1% formic acid in acetonitrile as organic phase on a BEH C18 column (2.1 mm × 150 mm, 3.5 µm, Waters, Dublin, Ireland). As a result, the BEH column offered the best separation, apparent peak shape and signal, and short analysis time (<20 min). Water and acetonitrile showed better performance than methanol for illicit compounds. Formic acid in water was preferred to enhance ionization of target compound during the electrospray process [16,17]. Chromatograms of the 64 compounds obtained from spiked dietary supplement samples with all compounds at the target concentrations are shown in Figure S1. Despite the many kinds of target analytes, the high-quality separation of 64 compounds was achieved. The gradient elution conditions were also optimized by the variation of mobile phases through the repeated analysis of the standard mixture. In addition, eluting the weakly retained compounds, it is necessary to raise the content of aqueous solvent during the gradient profile to increase of the resolution of the column [18,19]. The determination of target compounds was performed using ESI positive and negative ion modes through the direct infusion of standard solutions (100 µg L−1). We optimized the characteristic MS/MS parameters specifically for each analyte: capillary voltage of 3.5 kV; desolvation and source temperatures of 500 and 150 °C, respectively; desolvation gas flow of 650 L/h; and cone voltage of 30 V. Collision-induced dissociation was performed using argon. Collision energies (5–60 eV) were tested to give the maximum of intensity for the fragment ions. MRM transition with the most abundant ion was used for quantification, while the other two transitions were used for confirmation ions. In 45 compounds, it was found that singly charged precursor ions [M + H]+ were the most abundant, whereas the deprotonated ion [M − H]− was most abundant in 19 compounds (Table 1). MS parameters were optimized in both positive and negative ionization modes with variation of a single setting at a time and evaluation of the sensitivity. Dwell time has a huge influence on the quality of the mass spectra, since lower dwell time leads to more noise on the baseline the peaks. Thus, 16–129 ms of dwell time are desirable for better signal to noise and sensitivity. Due to the large number of substances to be analyzed, a method was developed for simultaneous analysis of 64 illicit compounds using a scheduled multiple reaction monitoring (s-MRM) algorithm monitor. The s-MRM improved the quality of the chromatograms, signal response, and enhanced signal to noise (S/N) ratio, resulting in more data points [20,21].

Table 1.

MRM transition and optimized parameters of LC-MS/MS for 64 target compounds.

Compounds Ion Mode (ESI +/−) Molecular Weight (g/mol) Precursor Ion (m/z) Product Ion (m/z) Collision Energy (eV) Cone Voltage (V) Retention Time (min)
2,4-Dinitrophenol
(2,4-DNP)		 − 184.1 183.1 109 25 30 9.46
137 20
153 15
7-keto-dehydroepiandrosterone
(7-keto-DHEA)		 + 302.4 303.3 81 24 30 9.72
267 15
285 15
Amphetamine + 135.2 136.1 41 26 20 4.98
91 15
119 10
Asarone + 208.3 209.1 151 24 30 13.3
179 22
194 16
Atropine + 289.4 290.1 77 35 20 6.37
93 28
124 22
Berberine + 336.4 336.2 292 28 25 8.63
306 28
320 25
beta-methylphenethylamine
(BMPEA)		 + 135.2 136.1 91 15 30 4.79
119 10
- -
Bisacodyl + 361.4 362.1 167 50 30 10.9
184 28
226 18
Buformin + 157.1 158.2 43 20 30 1.41
60 15
116 15
Cascaroside A − 580.1 579.1 268 52 35 5.89
297 38
459 20
Cascaroside B − 580.1 579.1 268 52 35 5.31
297 38
459 20
Cascaroside C − 563.8 563.2 251 64 30 7.23
281 40
443 24
Cascaroside D − 563.8 563.2 251 64 30 6.96
281 40
443 24
Chlorothiazide − 295.7 293.9 115 52 30 3.99
179 46
214 30
Cimifugin + 306.3 307.2 221 28 35 7.54
235 28
259 28
Dehydroepiandrosterone
(DHEA)		 + 288.4 289.2 197 18 30 12.5
213 18
253 12
Echinacoside − 786.7 785.3 133 80 40 6.53
161 46
623 36
Ephedrine + 165.2 166.2 117 20 35 3.51
133 20
148 13
Fenfluramine + 231.3 232.2 109 35 35 8.33
159 15
187 15
Fluoxetine + 309.3 310.1 44 10 30 10.6
117 50
148 10
Glibenclamide − 494.0 492.1 127 50 30 13.9
170 32
367 18
Gliclazide − 323.4 322.1 80 48 40 12.45
106 42
170 22
Glimepiride − 490.6 489.3 225 34 45 14.3
350 20
364 28
Glipizide − 445.5 444.1 154 54 30 11.4
170 34
319 20
Hesperidin + 610.2 611.2 153 52 30 8.02
303 22
449 12
Hydrastine + 383.4 384.2 190 20 35 7.32
293 25
323 20
Hydrochlorothiazide − 297.7 295.9 78 28 40 4.80
205 24
269 18
Hydroflumethiazide − 331.3 329.9 160 38 30 6.62
239 24
303 20
Icariin + 676.7 677.3 313 58 30 9.51
369 32
531 16
Icaritin + 368.4 369.1 135 35 30 15.8
187 40
313 23
Kavain + 230.3 231.1 115 14 35 11.9
153 22
185 15
Levodopa + 197.2 198.1 107 25 20 0.82
152 15
181 10
Levothyroxine + 776.9 777.6 324 54 25 10.7
351 46
605 40
Liothyronine + 651.0 651.7 197 68 30 9.94
225 42
479 34
Lorcaserine + 195.7 196.1 129 28 30 7.61
139 22
144 20
Lovastatin + 404.5 405.3 199 12 35 16.1
225 14
285 12
Magnoflorine + 342.4 342.2 192 35 30 6.13
265 25
298 25
Melatonin + 232.3 233.1 130 40 30 8.11
143 25
174 16
Methylchlorothiazide - 360.2 357.9 194 22 30 9.25
258 18
322 14
Mexamine
(5-methoxytryptamine1)		 + 190.2 191.1 130 38 30 5.29
159 22
174 16
N-nitrosofenfluramine + 260.3 261.1 109 44 30 14.1
159 22
187 12
Noopept + 318.4 319.2 70 22 35 9.20
188 14
216 10
Oxilofrine + 181.2 182.1 105 22 40 0.83
133 18
149 20
Oxindole + 133.2 134.1 51 35 45 6.87
79 22
106 16
Oxitriptan
(5-hydroxytryptohpane)		 + 220.2 221.1 106 32 30 0.83
134 24
160 24
Phendimetrazine + 191.3 192.2 74 18 30 5.26
117 21
146 24
Phenformin + 205.3 206.1 60 20 35 4.90
77 35
164 20
Phenolphtalein + 318.3 319.1 141 42 25 10.7
197 30
225 22
Phentermin + 149.2 149.9 55 18 30 5.86
65 34
133 10
Picamilon + 208.2 209.1 78 25 30 1.77
106 20
108 22
Rauwolscine
(α-yohimbine)		 + 354.4 355.2 117 40 30 7.49
144 32
212 22
Reserpine + 608.7 609.3 174 35 30 10.6
195 35
397 30
Salbutamol + 239.3 240.1 121 25 35 2.28
148 20
166 15
Salicin − 286.3 331.1 93 50 40 3.94
123 12
285 10
Salicylic acid - 138.1 136.9 65 26 45 8.44
93 25
- -
Scopolamine + 303.4 304.2 121 20 35 5.31
138 15
156 15
Sennoside A − 862.8 861.2 224 40 30 7.55
386 36
699 28
Sennoside B − 862.8 861.2 224 40 30 7.01
386 36
699 28
Serotonin
(5-hydroxytryptamine)		 + 176.2 177.1 105 26 50 1.43
115 24
142 22
Synephrine + 167.2 168.1 107 26 30 0.82
119 16
135 20
Tolbutamide + 270.4 271.1 74 12 55 11.5
91 30
155 16
Trichloromethiazide − 380.7 377.9 215 34 35 8.80
242 22
306 14
95 18
- -
Vinpocetine + 350.5 351.1 280 26 30 9.98
294 25
308 25
Yohimbine
(β-yohimbine)		 + 354.4 355.2 117 40 35 7.49
144 32
212 22
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The bold text expresses the quantification ion.

2.2. Method Validation

The LOQ, accuracy, and precision at three target concentrations are shown in Table 2. The linearity of calibration curves was evaluated by six-point calibrations of standards in solvents at the levels in the range of 0.5–200 µg L−1. The correlation coefficients (r2) were higher than 0.98 in all compounds. Our results show a good linearity that allowed coverage for all target compounds.

Table 2.

LOQ, accuracy, and precision at three target levels in dietary supplement.

Compounds Linearity
(r2)		 LOD
(ng kg−1)		 LOQ
(ng kg−1)		 Target Concentrations
(ng kg−1)		 Accuracy (% Recovery) Precision (% RSD)
Intra-Day Inter-Day Intra-Day Inter-Day
2,4-Dinitrophenol
(2,4-DNP)		 0.9965 2 6 10 91.4 92.7 5.41 2.67
50 107 102 0.83 4.01
100 96.4 100 1.68 3.82
7-keto-dehydroepiandrosterone
(7-keto-DHEA)		 0.9977 1 2 10 86.9 103 2.26 3.13
50 108 100 2.17 3.45
100 107 99.8 0.81 3.27
Amphetamine 0.9995 1 4 10 109 95.6 2.48 4.66
50 104 99.7 2.19 2.53
100 104 100 1.54 2.91
Asarone 0.9991 1 4 10 89.8 94.6 6.59 5.02
50 92.0 102 6.95 3.85
100 88.1 99.4 6.17 3.68
Atropine 0.9993 2 6 10 99.2 92.8 4.50 5.72
50 109 92.3 5.45 4.54
100 106 98.5 0.80 2.67
Berberine 0.9988 2 6 10 84.6 91.2 3.65 3.34
50 104 99.9 2.32 4.78
100 102 102 3.23 3.57
beta-methylphenethylamine
(BMPEA)		 0.9998 1 3 10 111 95.2 5.04 3.19
50 105 100 3.21 2.20
100 102 100 2.92 1.86
Bisacodyl 0.9978 0.3 1 10 89.2 101 1.76 1.32
50 96.5 92.9 0.94 2.05
100 98.9 92.3 1.92 1.62
Buformin 0.9965 2 6 10 87.4 86.4 1.58 4.94
50 111 103 2.20 2.57
100 114 97.9 1.29 2.76
Cascaroside
A, B, C, D		 0.9991 3 9 10 86.9 100 8.02 3.69
50 100 101 1.13 3.33
100 100 98.9 1.35 2.64
Chlorothiazide 0.9962 3 8 10 95.1 88.6 2.68 4.52
50 102 102 2.14 4.86
100 103 102 1.51 2.12
Cimifugin 0.9994 1 4 10 94.1 88.9 3.16 6.29
50 102 102 2.83 4.74
100 102 96.6 4.22 2.58
Dehydroepiandrosterone
(DHEA)		 0.9997 1 5 10 95.2 99.3 6.90 5.86
50 101 97.1 6.12 3.88
100 89.1 98.1 2.57 5.75
Echinacoside 0.9993 3 9 10 101 95.8 7.55 6.73
50 94.5 96 7.33 6.39
100 93.9 103 3.53 4.37
Ephedrine 0.9975 1 4 10 96.6 90.5 1.33 3.24
50 103 99.3 1.64 1.81
100 106 100 1.34 2.62
Fenfluramine 0.9984 2 6 10 91.8 91.1 2.69 4.68
50 104 105 8.25 3.18
100 103 101 3.72 2.75
Fluoxetine 0.9992 1 5 10 95.2 91.4 5.55 2.46
50 114 102 1.57 2.32
100 110 103 1.35 1.98
Glibenclamide 0.9994 2 6 10 89.0 95.9 3.63 2.75
50 104 97.3 1.95 1.28
100 103 96.6 1.15 2.59
Gliclazide 0.9994 1 3 10 84.4 98.5 2.25 2.78
50 101 97.8 0.98 2.79
100 99.8 96.2 1.39 2.45
Glimepiride 0.9997 1 2 10 89.3 99.3 1.54 1.88
50 102 100 0.98 1.77
100 101 98.3 1.65 2.12
Glipizide 0.9991 1 4 10 80.1 94.7 1.09 3.17
50 101 98.7 1.26 2.27
100 101 96.8 1.35 3.05
Hesperidin 0.9999 1 3 10 97.5 97.6 4.01 5.31
50 104 100 1.34 3.33
100 103 101 2.43 1.74
Hydrastine 0.9980 1 4 10 91.3 87.3 8.54 4.63
50 107 96.9 4.00 4.23
100 106 99.8 4.18 3.25
Hydrochlorothiazide 0.9989 2 8 10 95.4 98.8 7.53 5.47
50 98.9 97.5 4.37 2.89
100 99.7 100 6.67 1.58
Hydroflumethiazide 0.9989 2 7 10 97.4 92.5 6.21 7.34
50 107 92.7 6.09 4.66
100 112 94.7 6.51 2.23
Icariin 0.9984 1 4 10 85.1 92 1.68 2.01
50 105 98.9 1.12 2.64
100 106 100 1.39 2.57
Icaritin 0.9985 1 3 10 99.3 89.3 5.97 2.58
50 106 102 2.81 3.31
100 101 102 1.77 1.61
Kavain 0.9994 1 3 10 84.1 85 2.15 3.02
50 104 103 2.07 1.32
100 99.5 100 1.32 1.09
Levodopa 0.9996 1 5 10 86.6 92.6 2.74 4.85
50 98.8 102 1.17 2.41
100 97.2 103 1.55 1.18
Levothyroxine 0.9997 1 5 10 96.1 100 3.97 3.33
50 101 100 2.63 3.70
100 103 98.8 4.33 2.31
Liothyronine 0.9999 2 5 10 96.9 95 2.50 3.15
50 104 98.3 0.99 4.30
100 104 98.9 2.16 2.25
Lorcaserine 0.9979 1 4 10 85.0 92.9 2.70 3.93
50 105 102 3.75 3.41
100 107 103 1.98 3.17
Lovastatin 0.9989 1 3 10 88.4 91.5 2.22 2.50
50 105 108 3.60 1.51
100 98.7 108 3.96 0.93
Magnoflorine 0.9985 2 8 10 99.3 94.4 5.97 6.03
50 106 107 2.81 2.92
100 101 102 1.77 2.61
Melatonin 0.9994 1 2 10 91.3 81.9 3.71 4.23
50 104 102 1.61 2.36
100 99.2 100 2.13 2.46
Methylchlorothiazide 0.9981 2 8 10 81.2 96.8 5.59 6.42
50 103 99.1 3.77 5.84
100 100 99.2 1.93 3.73
Mexamine
(5-methoxytryptamine1)		 0.9960 1 5 10 95.7 99.4 9.15 6.53
50 112 102 4.23 4.75
100 108 105 1.72 3.85
N-nitrosofenfluramine 0.9989 1 5 10 83.1 88 2.89 3.15
50 105 101 2.51 2.63
100 103 99 1.03 2.78
Noopept 0.9996 1 4 10 83.9 85.6 2.32 3.03
50 101 98.3 1.66 4.59
100 101 98.1 1.72 4.31
Oxilofrine 0.9988 1 5 10 97.4 93 2.15 3.29
50 111 106 5.04 2.67
100 112 98.1 3.11 1.98
Oxindole 0.9997 1 2 10 102 90 2.76 2.96
50 102 97.9 2.10 3.41
100 103 99.3 1.34 1.64
Oxitriptan
(5-hydroxytryptohpane)		 0.9991 2 8 10 97.2 85.9 2.83 4.69
50 102 98 2.56 3.33
100 105 100 2.99 1.77
Phendimetrazine 0.9987 1 5 10 91.0 89.4 2.37 4.90
50 103 95.8 2.64 3.06
100 100 99.1 2.66 1.93
Phenformin 0.9976 1 4 10 88.6 83 7.17 7.18
50 110 98.1 5.41 3.52
100 106 101 3.57 3.52
Phenolphtalein 0.9993 1 5 10 90.4 86.2 3.09 3.42
50 103 100 1.21 2.12
100 99.0 99.3 3.51 2.95
Phentermin 0.9992 1 4 10 101 85.9 3.02 4.84
50 104 100 1.30 3.28
100 105 100 2.50 3.11
Picamilon 0.9992 1 4 10 98.9 85.5 1.99 4.03
50 102 97.5 1.23 2.55
100 103 97.5 0.78 1.84
Rauwolscine
(α-yohimbine)		 0.9989 0.3 1 10 103 91.9 4.08 5.98
50 102 96.6 5.00 3.15
100 91.6 99.1 4.57 4.34
Reserpine 0.9997 1 5 10 99.2 97.5 2.14 2.10
50 102 100 2.13 3.23
100 102 99.1 1.67 3.18
Salbutamol 0.9981 2 6 10 87.3 85.5 1.97 3.18
50 107 100 2.42 2.61
100 109 100 1.05 1.25
Salicin 0.9994 5 10 10 97.8 92 5.41 4.61
50 100 95.4 1.91 4.72
100 102 94.1 4.49 4.24
Salicylic acid 0.9987 3 8 10 90.3 98.1 8.22 4.85
50 98.1 95.5 4.50 4.97
100 97.2 98.8 4.82 6.85
Scopolamine 0.9966 3 6 10 92.0 87.5 2.80 8.09
50 110 99.9 4.76 4.17
100 108 101 4.68 2.47
Sennoside
A, B		 0.9943 3 8 10 78.5 98.9 4.83 3.74
50 98.4 100 4.69 5.00
100 102 98.9 4.58 4.54
Serotonin
(5-hydroxytryptamine)		 0.9937 3 9 10 88.0 86.3 6.02 6.58
50 108 101 5.32 4.40
100 108 104 2.39 2.78
Synephrine 0.9970 3 6 10 92.5 94.6 6.14 7.59
50 112 102 3.97 3.48
100 95.6 93.6 3.44 3.34
Tolbutamide 0.9997 1 4 10 92.3 94.1 2.90 2.23
50 103 100 2.30 1.83
100 103 100 1.92 1.49
Trichloromethiazide 0.9973 3 8 10 83.1 91.5 5.15 7.52
50 104 97.4 7.41 2.38
100 102 96.3 2.80 1.92
Vinpocetine 0.9994 3 3 10 92.0 87.9 2.17 3.85
50 110 100 2.10 2.10
100 109 101 1.00 3.03
Yohimbine
(β-yohimbine)		 0.9989 0.3 1 10 103 91.9 4.08 5.98
50 102 96.7 5.00 3.15
100 91.6 99.2 4.57 4.34
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The accuracy (expressed as recovery, percent) and precision (expressed as RSD, percent) of target compounds were evaluated in spiked blank samples at three target concentrations (10, 50, and 100 µg L−1). The overall recoveries for all target compound ranged 78.5–114%. The precision ranged 0.78–9.15% in intra-day, inter-day, and inter-lab validation processes. These results indicate the good precision and reliability of the proposed method. The LODs and LOQs were calculated by three and ten times the standard deviation divided by the slope of standard curves, respectively. The LOD and LOQ values were <5 µg L−1 and <10 µg L−1, respectively. Inter-laboratory validation test was conducted to evaluate the ruggedness with three institutions. As a result, recovery ranged 61.1–132% with RSD less than 15% for all target compounds. The analytical method showed an acceptable accuracy and precision, with satisfactory values for all method validation parameters according to the requirements of the AOAC guidelines.

Most of collected samples were capsules, powders, tablets, and soft-gels (Table S3). However, herbal supplements may have a complex matrix that can potentially interfere with the determination of target compounds and cause matrix effect in mass analysis [10]. The matrix effect should be evaluated for further method development and for a variety of plant food and herbal supplement samples.

2.3. Application of Real Samples

The proposed method was applied in dietary supplement samples (n = 200) randomly collected from 10 websites. The product category (indication for which product was marketed) of samples was classified into sexual performance (15.5%), weight loss (47%), muscle strengthening enhancement (19%), relaxation (5.5%), and others (13%). The type of samples was categorized into capsules (51%), powders (25%), tablets (10.5%), soft-gels (6%), liquids (5.5%), tea bag (0.5%), and energy bars (1%) (Table S3). The detection numbers and concentrations in collected dietary supplements are described in Table 3. The total detection rate was 13.5% (27 out of 200 samples). Eleven kinds of compounds were detected in 27 samples. In weight loss products, 16 samples were detected in 94 samples (17%). For muscular strengthening products, seven samples were detected in 38 samples (18.4%). In relaxing products, six samples were detected in 11 samples (54.5%). For sexual enhancement products, four samples detected in 31 samples (12.9%). Moreover, the combination of two or more compounds in one sample was detected in four samples. Examples of chromatogram and mass spectra are shown in Figure S2.

Table 3.

Detected numbers and concentrations in dietary supplements by product category.

Product Category by Labelling
(Sample Number)		 Compounds Detected Number Detected
Sample No.		 Concentrations Contents
Mean
(mg g−1)		 Range
(mg g−1)		 Contents per Unit
(mg/Unit)
Sexual enhancement products
(n = 31)		 DHEA 1 S-3 39.7 21.4
Icariin 2 S-21), S-88 1.90 0.77–3.03 1.34–2.12
Magnoflorine 1 S-21) 0.54 0.38
Weight loss products
(n = 94)		 Cascarosides 3 S-4, S-77, S-79 8.02 5.77–9.62 1.47–6.16
Melatonin 1 S-171 3.35 4.02
Mexamine 1 S-71) 3.64 4.14
Oxitriptan (5-HTP) 2 S-71), S-172 20.4 3.50–37.2 3.99–23.1
Sennosides 2 S-161, S-162 24.5 13.9–35.0 9.89–19.6
Synephrine 5 S-6, S-71), S-10, S-13, S-140 27.2 3.70–75.7 2.92–67.4
Yohimbine 2 S-71), S-60 2.32 1.69–2.94 1.03–1.47
Muscular strengthening products
(n = 38)		 Synephrine 3 S-61, S-107, S-1551) 33.3 14.9–56.5 18.3–134
Yohimbine 4 S-9, S-16, S-142, S-1551) 4.60 0.51–11.0 1.78–9.31
Relaxing products
(n = 11)		 Kavain 1 S-73 18.8 18.8 14.3
Melatonin 1 S-481) 6.74 1.94
Oxitriptan (5-HTP) 4 S-17, S-22, S-481), S-82 115 56.6–226 20.6–140
Others
(n = 26)
Total
(n = 200)		 11 compounds 33 27 samples 0.51–226 0.38–140
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Synephrine was the most frequently detected in dietary supplements (eight samples), and the concentrations ranged 3.70–75.7 mg g−1. Synephrine is a substance that occurs naturally in plants and is added to dietary supplements for weight-loss and sports performance [22]. However, synephrine is prohibited because it may have adverse effect on human health. Synephrine has structural and pharmacological similarities with ephedrine; therefore, it can cause similar side effects. Furthermore, synephrine and DHEA were added to the inspection program in competitions of the World Anti-Doping Agency [22].

Yohimbine was detected in six samples at concentrations of 0.51–11.0 mg g−1. A similar concentration (6.6 mg g−1) of yohimbine was detected in one sample [23]. In Korea, yohimbine is a drug used in veterinary medicine in livestock products based on maximum residue limit [24]. Although drugs of yohimbine are no longer prescribed by US physicians, yohimbine remains an ingredient in dietary supplements for sexual and sports enhancements. Dietary supplements containing yohimbine can pose a significant risk to consumers [25]. Yohimbine is a natural tryptamine alkaloid extracted from the bark of various plants. Due to the side effects, this substance in sexual enhancement products carries a psychological health risk [3].

5-Hydroxytryptophan (5-HTP) known as oxitriptan was detected in the range of 3.50–226 mg g−1 in six samples. 5-HTP is a naturally occurring amino acid and chemical precursor as well as a metabolic intermediate in serotonin synthesis. In certain countries, 5-HTP is added in dietary supplements to treat depression and insomnia [26]. Melatonin was detected in two samples (3.35–6.74 mg g−1). Melatonin has been sold as on over-the-counter drug and is not regulated by the Food and Drug Administration, whereas, in Europe and Asia-Pacific, it is a prescription drug. 5-HTP and melatonin may cause side effects such as delirium, risk of falling down, and sleep-related eating disorder.

Cascarosides were detected in three samples (5.77–9.62 mg g−1). Sennosides were detected in two samples (13.9–35.0 mg g−1). In a previous study, sennoside A and B were detected in the range of 0.41–3.27 mg g−1 in 16 samples. Cascara and senna extracts are well known effective laxative drugs and used in the treatment of intestinal constipation. Some side effects have been reported in the administration of senna extracts [27]. Icariin was detected in two samples (0.77–3.03 mg g−1). In a previous study, icariin was detected in 19 samples at 0.1–19.0 mg g−1 in adulterated products [23]. Kavain (18.8 mg g−1) was detected in one sample. These findings suggest that plant origin dietary supplements are commonly used to promote sexual health and weight loss. Many herbal supplements have potential benefits, however the health risks of various phytotherapeutic compounds need to be elucidated.

Overall, illicit compounds continue to be detected in dietary supplements for sexual enhancement and weight loss. The pharmaceuticals in dietary supplements have potential health risks due to misuse, over use, or interaction with other pharmaceuticals in drugs and supplements. The adverse effects reported for the use of dietary supplements include stroke, acute liver injury, kidney failure, pulmonary embolisms, and death [4]. Further studies are needed to improve the wide coverage of sampling including websites and more private ways of social networking.

3. Materials and Methods

3.1. Chemical and Reagents

Psychoactive substances (amphetamine, 99.9%; atropine, 97.0% BMPEA, 95.0%; fenfluramine hydrochloride, 99.2%; lorcaserin hydrochloride, 99.0%; phendimetrazine tartrate, 99.9%; phentermine hydrochloride, 99.9%; and salicylic acid, 99.9%) were obtained from the Ministry of Food and Drug Safety (Cheongju, South Korea) in accordance with Narcotics Control Act. In addition, synthetic compounds (7-keto-DHEA, 99.9%; cascaroside A and B, 98.0%; cascaroside C, 97%; cascaroside, D 99%; cimifugin, 99.0%; icaritin, 99.0%; n-nitrosofenfluramine, 99.0%; salicin, 99.0%; and synephrine, 99%) were synthesized by the Ministry of Food and Drug Safety (Cheongju, South Korea) for testing. Other compounds of high purity grade (≥95%) were purchased from Wako Pure Chemical Industries Inc. (Osaka, Japan), USP (Rockville, MD, USA), or Sigma-Aldrich (Buchs, Switzerland). The manufacturer and purity for 64 chemical standards are presented in Table S2. All reagents and solvents (analytical grade) were purchased from Merck Inc. (Darmstadt, Germany) or Sigma-Aldrich (Buchs, Switzerland). The samples were purified by filtration through polytetrafluoroethylene (PTFE) filter provided by Teknokroma (Barcelona, Spain). Individual standard stock solution was prepared by dissolving each standard in methanol with a concentration of 1000 µg mL−1 and were stored at −4 °C. Working mix standard solutions containing all compounds were daily prepared by appropriate dilution of the stock standard solution with 70% methanol.

3.2. Sample Preparation

The dietary supplements in the form of capsules, tablets, powder, oral liquids, and soft-gels were obtained through overseas direct purchase. All tablets were homogenized with a blender. The shells of all of the capsules and soft-gels were removed to mix the powder inside. First, 1.0 ± 0.01 g sample was transferred into a 50-mL volumetric flask. Each sample (1 g) was accurately weighed, and then mixed in water (15 mL) for 1 min. After that, methanol was added (20 mL), and the samples sonicated for 20 min to extract the analytes. Methanol was added into the final volume up to 50 mL. The sample extracts were filtered through a polytetrafluoroethylene (PTFE) syringe filter with 0.22-μm pore size before LC-MS/MS analysis.

3.3. LC-MS/MS Analysis

LC-MS/MS analysis was performed on a Waters Acquity I-Class UPLC system (Waters, Milford, MA, USA) coupled with a Xevo TQ-S (Triple Quadrupole Mass Spectrometer) equipped with an electrospray ionization source (Waters, Milford, MA, USA). The chromatographic separation was carried out using Acquity BEH C18 column (2.1 mm × 150 mm, 3.5 µm, Waters, Dublin, Ireland). The mobile phase was made up of Solvent A (0.1% formic acid in water), and Solvent B (0.1% formic acid in acetonitrile). The oven and column temperature were maintained at 40 °C, and the flow rate was set at 0.3 mL min−1. The optimized gradient was as follows: 0–3 min, 5% B; 3–18 min, 80% B; 18–18.1 min, 100% B; 18.1–20.9 min, 100% B; 20.9–21 min 5% B; and 21–25 min, 5%. The injection volume was 5 µL. Mass spectrometer conditions were set as follows: the capillary voltage, 3.5 kV in ESI positive mode and −2.8 kV in the ESI negative mode. The desolvation and source temperatures were set at 500 and 150 °C, respectively. The cone and desolvation gas flow rates (nitrogen) were 60 and 600 L h−1, respectively. Collision-induced dissociation was performed using argon as the collision gas at a pressure of 4 × 10−3 mbar in the collision cell. Data collection was implemented in multiple reaction monitoring (MRM) mode using the MassLynx software (Waters, UK). The [M + H]+ and [M–H]– ions were selected as precursor ions; the two or three most intense product ions were used as product ions. Table 1 describes the optimized mass parameters, MRM transition, and retention times of the 64 illicit compounds.

3.4. Method Validation

The method validation procedure was performed through estimation of the linearity, limit of detection (LOD), limit of quantitation (LOQ), accuracy, and precision based on AOAC guideline (AOAC, 2016). Calibration curves were obtained by linear regression analysis over the target concentration levels in the range of 0.5–200 µg L−1, depending on the compounds. The LODs and LOQs were calculated by three and ten times the standard deviation divided by the slope of standard curves, respectively. The accuracy (recovery, percent) and precision (relative standard deviation, percent) were determined through the analysis of negative sample of dietary supplements spiked at 10, 50, and 100 µg kg−1 (bisacodyl and yohimbine were tested at 1, 5, and 10 µg kg−1) in five replicates for 64 illicit compounds within a single day. The intra- and inter-day precision were also determined in triplicate within one day and over three consecutive days, respectively.

4. Conclusions

In this study, we reported the development and optimization multi-class determination of 64 selected compounds in dietary supplements using LC-MS/MS. The present method showed suitable linearity, accuracy, precision, LOD, and LOQ to detect the multiple classes of 64 illicit compounds in dietary supplements. The detection rate was 13.5% (27 out of 200 samples). Eleven compounds were detected in 27 dietary supplement samples. Synephrine and yohimbine were the most detected compounds in weight loss and muscular strengthening products. The proposed method was successfully validated and applied for identification and confirmation of illicit compounds in dietary supplements. As the dietary supplement industry continues to grow in the world, adulterated products should be regularly monitored to protect public from potential health risks.

Supplementary Materials

The following are available online, Figure S1: Chromatogram of 64 compounds at 0.05 mg L−1; Figure S2: Example of chromatogram and mass spectra in sample S-162 (sennosides); Table S1: Selection criteria of 64 target compounds based on Korean legislation; Table S2: The manufacturer and purity on target compounds; Table S3: The collected sample numbers based on sample type and product category.

Click here for additional data file. (831KB, pdf)

Author Contributions

D.S., experimental work and drafting of the manuscript; H.-S.K., study design and writing and revising of the manuscript; H.K., revising of the manuscript; and G.M., reviewing of the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This study was supported by grants (Nos. 17161MFDS062 and 20161MFDS052) from the Ministry of Food and Drug Safety of Korea from 2017 to 2020.

Conflicts of Interest

The authors declare no conflict of interest.

References

  • 1.Patterson R., Mabe P., Mitchell E.N., Cory W. Lifestyle illicit drug seizures: A routine ESI-LC-MS method for the identification of sildenafil and vardenafil. Forensic Sci. Int. 2012;222:83–88. doi: 10.1016/j.forsciint.2012.05.003. [DOI] [PubMed] [Google Scholar]
  • 2.Rocha T., Amaral J.S., Oliveira M.B.P.P. Adulteration of Dietary Supplements by the Illegal Addition of Synthetic Drugs: A Review. Compr. Rev. Food Sci. Food Saf. 2016;15:43–62. doi: 10.1111/1541-4337.12173. [DOI] [PubMed] [Google Scholar]
  • 3.Corazza O., Martinotti G., Santacroce R., Chillemi E., Di Giannantonio M., Schifano F., Cellek S. Sexual Enhancement Products for Sale Online: Raising Awareness of the Psychoactive Effects of Yohimbine, Maca, Horny Goat Weed, and Ginkgo biloba. BioMed Res. Int. 2014;2014:841798. doi: 10.1155/2014/841798. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Tucker J., Fischer T., Upjohn L., Mazzera D., Kumar M. Unapproved Pharmaceutical Ingredients Included in Dietary Supplements Associated with US Food and Drug Administration Warnings. JAMA Netw. Open. 2018;1:e183337. doi: 10.1001/jamanetworkopen.2018.3337. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Lee E.S., Lee J.H., Han K.M., Kim J.W., Hwang I.S., Cho S., Han S.Y., Kim J. Simultaneous determination of 38 phosphodiestrase-5 inhibitors in illicit erectile dysfunction products by liquid chromatography-electrospray ionization-tandem mass spectrometry. J. Pharm. Biomed. Anal. 2013;83:171–178. doi: 10.1016/j.jpba.2013.05.009. [DOI] [PubMed] [Google Scholar]
  • 6.Lee J.H., Kim N.S., Han K.M., Kim S.H., Cho S., Kim W.S. Monitoring by LC-MS/MS of 48 compounds of sildenafil, tadalafil, vardenafil and their analogues in illicit health food products in the Korean market advertised as enhancing male sexual performance. Food Addit. Contam. Part A Chem. Anal. Control Expo. Risk Assess. 2013;30:1849–1857. doi: 10.1080/19440049.2013.826878. [DOI] [PubMed] [Google Scholar]
  • 7.Kim H.J., Lee J.H., Park H.J., Cho S.H., Cho S., Kim W.S. Monitoring of 29 weight loss compounds in foods and dietary supplements by LC-MS/MS. Food Addit. Contam. Part A Chem. Anal. Control Expo. Risk Assess. 2014;31:777–783. doi: 10.1080/19440049.2014.888497. [DOI] [PubMed] [Google Scholar]
  • 8.Shin D., Kang H.-S., Kim H.-S., Moon G. Determination of 11 Illicit Compounds in Dietary Supplements Using High-Performance Liquid Chromatography and Liquid Chromatography-Tandem Mass Spectrometry. J. Food Hyg. Saf. 2020;35:326–333. doi: 10.13103/JFHS.2020.35.4.326. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Lee J.H., Park H.N., Kim N.S., Park S., Bogonda G., Oh K., Kang H. Application of screening methods for weight-loss compounds and identification of new impurities in counterfeit drugs. Forensic Sci. Int. 2019;303:109932. doi: 10.1016/j.forsciint.2019.109932. [DOI] [PubMed] [Google Scholar]
  • 10.Paíga P., Rodrigues M.J.E., Correia M., Amaral J.S., Oliveira M.B.P.P., Delerue-Matos C. Analysis of pharmaceutical adulterants in plant food supplements by UHPLC-MS/MS. Eur. J. Pharm. Sci. 2017;99:219–227. doi: 10.1016/j.ejps.2016.12.024. [DOI] [PubMed] [Google Scholar]
  • 11.Kim E.H., Seo H.S., Ki N.Y., Park N.H., Lee W., Do J.A., Park S., Baek S.Y., Moon B., Oh H.B., et al. Reliable screening and confirmation of 156 multi-class illegal adulterants in dietary supplements based on extracted common ion chromatograms by ultra-high-performance liquid chromatography-quadrupole/time of flight-mass spectrometry. J. Chromatogr. A. 2017;1491:43–56. doi: 10.1016/j.chroma.2017.02.032. [DOI] [PubMed] [Google Scholar]
  • 12.Guo C., Niu C., Zhou L., Wang W., Nie Y., Liu Q., Zhang L., Chen Z., Wang W., Xu Y. Targeted and nontargeted screening and identification of 50 antihypertensive adulterants in dietary supplements and herbal medicines using quadrupole–orbitrap high resolution mass spectrometry with compound database. J. Sep. Sci. 2020 doi: 10.1002/jssc.201901121. [DOI] [PubMed] [Google Scholar]
  • 13.Cho S.H., Park H.J., Lee J.H., Kim H.J., Cho S., Yoon C.Y., Kim W.S. Monitoring of 35 illegally added steroid compounds in foods and dietary supplements. Food Addit. Contam. Part A Chem. Anal. Control Expo. Risk Assess. 2014;31:1470–1475. doi: 10.1080/19440049.2014.946100. [DOI] [PubMed] [Google Scholar]
  • 14.Hur J., Lee W., Kim B.H., Kim H.J., Lee D.H., Lee J.H., Lee Y.M., Oh H.B., Hong J. Comprehensive screening of multiclass illegal adulterants in herbal supplements and Spice-type drugs using specific MS/MS fragmentations by UHPLC-Q/TOF-MS. Anal. Methods. 2019;11:5260–5273. doi: 10.1039/c9ay01517e. [DOI] [Google Scholar]
  • 15.Burrai L., Nieddu M., Carta A., Trignano C., Sanna R., Boatto G. Validated LC-MS-MS Method for Multiresidual Analysis of 13 Illicit Phenethylamines in Amniotic Fluid. J. Anal. Toxicol. 2016;40:194–200. doi: 10.1093/jat/bkv143. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Shin D., Kang H.-S., Jeong J., Kim J., Choe W.J., Lee K.S., Rhee G.-S. Multi-residue Determination of Veterinary Drugs in Fishery Products Using Liquid Chromatography-Tandem Mass Spectrometry. Food Anal. Methods. 2018;11:1815–1831. doi: 10.1007/s12161-018-1179-0. [DOI] [Google Scholar]
  • 17.Kiontke A., Oliveira-Birkmeier A., Opitz A., Birkemeyer C. Electrospray Ionization Efficiency Is Dependent on Different Molecular Descriptors with Respect to Solvent pH and Instrumental Configuration. PLoS ONE. 2016;11:e0167502. doi: 10.1371/journal.pone.0167502. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Gritti F., Gilar M., Walter T.H., Wyndham K. Retention loss of reversed-phase chromatographic columns using 100% aqueous mobile phases from fundamental insights to best practice. J. Chromatogr. A. 2020;1612:460662. doi: 10.1016/j.chroma.2019.460662. [DOI] [PubMed] [Google Scholar]
  • 19.Feng J.H., Wei K.Z., Gao J.P., Xu X. Determination of adenosine phosphates in mouse myocardium tissue by HPLC with UV detection and using porous graphite carbon column. J. Chromatogr. B Anal. Technol. Biomed. Life Sci. 2020;1145:122110. doi: 10.1016/j.jchromb.2020.122110. [DOI] [PubMed] [Google Scholar]
  • 20.Voigt A.M., Skutlarek D., Timm C., Schreiber C., Felder C., Exner M., Faerber H.A. Liquid chromatography-tandem mass spectrometry as a fast and simple method for the determination of several antibiotics in different aqueous matrices. Environ. Chem. 2020;17:54. doi: 10.1071/EN19115. [DOI] [Google Scholar]
  • 21.Sutcharitchan C., Miao S., Li W., Liu J., Zhou H., Ma Y., Ji S., Cui Y. High performance liquid chromatography-tandem mass spectrometry method for residue determination of 39 plant growth regulators in root and rhizome Chinese herbs. Food Chem. 2020;322:126766. doi: 10.1016/j.foodchem.2020.126766. [DOI] [PubMed] [Google Scholar]
  • 22.Rossato L.G., Costa V.M., Limberger R.P., De Lourdes Bastos M., Remião F. Synephrine: From trace concentrations to massive consumption in weight-loss. Food Chem. Toxicol. 2011;49:8–16. doi: 10.1016/j.fct.2010.11.007. [DOI] [PubMed] [Google Scholar]
  • 23.Lee J.H., Park H.N., Park O.R., Kim N.S., Park S.K., Kang H. Screening of illegal sexual enhancement supplements and counterfeit drugs sold in the online and offline markets between 2014 and 2017. Forensic Sci. Int. 2019;298:10–19. doi: 10.1016/j.forsciint.2019.02.014. [DOI] [PubMed] [Google Scholar]
  • 24.Kang H.S., Kwon N.J., Jeong J., Lee K., Lee H. Web-based Korean maximum residue limit evaluation tools: An applied example of maximum residue limit evaluation for trichlorfon in fishery products. Environ. Sci. Pollut. Res. 2019;26:7284–7299. doi: 10.1007/s11356-019-04314-y. [DOI] [PubMed] [Google Scholar]
  • 25.Cohen P.A., Wang Y., Maller G., DeSouza R., Khan I.A. Pharmaceutical quantities of yohimbine found in dietary supplements in the USA. Drug Test. Anal. 2016;8:357–369. doi: 10.1002/dta.1849. [DOI] [PubMed] [Google Scholar]
  • 26.Yang T.H., Hsu P.Y., Meng M., Su C.C. Supplement of 5-hydroxytryptophan before induction suppresses inflammation and collagen-induced arthritis. Arthritis Res. Ther. 2015;17:364. doi: 10.1186/s13075-015-0884-y. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Cao Y., He Y., Wei C., Li J., Qu L., Zhang H., Cheng Y., Qiao B. Aquaporins Alteration Profiles Revealed Different Actions of Senna, Sennosides, and Sennoside A in Diarrhea-Rats. Int. J. Mol. Sci. 2018;19:3210. doi: 10.3390/ijms19103210. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

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Supplementary Materials

Click here for additional data file. (831KB, pdf)

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