A R T I C L E
LC–MS/MS monitoring of 22 illegal antihistamine compounds
in health food products from the Korean market
Jung Yeon Kim•Ji Yeon Choi• Chang Yong Yoon•
Sooyeul Cho•Woo Seong Kim •Jung Ah Do
Received: 18 August 2014 / Accepted: 5 December 2014 / Published online: 14 February 2015 Ó The Korean Society for Applied Biological Chemistry 2015
Abstract With the increasing popularity of dietary sup-plements, a critical analysis of safety issues concerning their use has become imperative. Despite several regulations and laws being in place, there have been several instances of adulterated health food-induced accidents. Therefore, in light of the growing seriousness of this problem, we at-tempted to detect the presence of 22 antihistamines in health foods sold in South Korea. 117 samples, representative of the various types of health foods, were screened by liquid chromatography with electrospray ionization tandem mass spectrometry (LC–ESI–MS/MS). The limit of detections (LODs) and limit of quantifications (LOQs) of the instrument ranged from 0.0003 to 0.3 lg/mL and from 0.0009 to 0.6 lg/ mL, respectively. The LODs and LOQs of the method ranged from 0.006 to 6.0 lg/mL and from 0.018 to 12.0 lg/mL, respectively. The calibration curve was linear with R2 be-tween 0.997 and 0.999. The mean recovery efficiency ranged from 89.7 to 111.8 %. On applying our method to screen 117 commercially available samples, we found that 116 samples were not detected targeted compounds and one sample contained diphenhydramine component.
Keywords Adulterant Antihistamine Health foods Liquid chromatography Mass spectrometry
Introduction
Herbal medicines and health foods currently play an im-portant role in health care, therapy, and prevention of disease all around the world (Bouldin et al. 1999). Health foods are becoming increasingly popular in the health and food industries, as globally, the tertiary function of foods (physiological regulation) is being preferred over its pri-mary and secondary functions (Hel et al. 2006). The worldwide market for health functional foods has been growing by 6.6 % each year; in the USA, it has grown by 7.7 % annually. In addition, the Japan market for health food has also seen a noticeable rise (Anon2010). In 2011, the total sales of domestic health foods in Korea were 1,368.2 billion won, of which, 1,312.6 billion was from domestic sales and 55.6 billion was from export sales. Red ginseng products accounted for 52.6 % of total sales, fol-lowed by vitamin and mineral products, individually au-thorized products, aloe products, and omega-3 products (KFDA 2012).
These products are often considered by many as being harmless because of their natural origin. They are also regarded as being useful in treating some chronic diseases and in the overall maintenance of human health. Their share in the global pharmaceutical market is notable and increases annually (Chan et al.1993). Unfortunately, some manufacturers tend to include synthetic drugs in formula-tions marketed as ‘herbal medicine’ or ‘dietary supple-ment’ in order to enhance the efficacy of their products (Chan and Critchley 1996). Dietary supplements are easy targets for adulteration with drug compounds. This is be-cause, in most countries, dietary supplements are more
Jung Yeon Kim and Ji Yeon Choi have contributed equally to this work.
J. Y. Kim J. Y. Choi C. Y. Yoon S. Cho W. S. Kim J. A. Do (&)
Advanced Analysis Team, National Institute of Food and Drug Safety Evaluation, Ministry of Food & Drug Safety, Osong
of ‘‘purely natural substances’’ (implying totally harmless substances, as declared by some manufacturers).
However, such an act is a violation of regulations and laws of most countries; in addition, the safety of those adulterated products has not been clinically tested, owing to which, the health of a consumer may be affected unpredictably. For example, a dietary supplement, a brewer’s yeast known as MEGATON, was imported into Incheon airport in South Korea from the USA in capsular form. It was found to contain a synthetic analog of vardenafil and acetyl vardenafil (Lee et al.2011), which are drugs used to treat erectile dysfunc-tion. In 2013, the Seoul special judicial police found an aphrodisiac adulterated with chlorpheniramine, an antihis-tamine that can cause dizziness, lowering of blood pressure, confusion, incoordination, and drowsiness. Antihistamines are also associated with other side effects such as weight gain, altered taste, headaches, and dry mouth. These side effects are likely to be used in various crimes. Various weight loss drugs (Deconinck et al. 2012; Rebiere et al. 2012), erectile dysfunction drugs (Toomey et al.2012), synthetic steroids (Ku et al. 1999), non-steroidal anti-inflammatory drugs (Bogusz et al.2006), and their analogs are often added illegally into many dietary supplements.
Illegal pharmaceuticals, including promethazine, chlormethiazole, chlorpheniramine, diclofenac, chlor-diazepoxide, hydrochlorothiazide, triamterene, diphenhy-dramine, and sildenafil, were found in randomly purchased Chinese/patent medicines from Chinatown retail stores in New York City. In particular, many anti-hypertensive pills, anti-inflammation pills, and eczema creams contained the antihistamine compounds, promethazine, chlorpheni-ramine, and diphenhydchlorpheni-ramine, respectively (Miller and Stripp 2007). These results suggest that dietary supple-ments, in particular Chinese herbal/patent medicines, are not regulated for purity or safety. Therefore, in order to ban the production and marketing of adulterated products, for safeguarding of human health and safety, drug adminis-tration bodies are in urgent need of a general, rapid, and effective method for screening adulterated herbal medici-nes and health foods appearing on the market.
Many recent studies have used methods such as HPLC, GC–MS, and LC–MS for the simultaneous analysis of an-tihistamine compounds. Gergov et al. (2001) reported the simultaneous screening of 18 antihistamine compounds in blood by liquid chromatography–ionspray tandem mass spectrometry, and Hasegawa et al. (2006) reported the si-multaneous determination of 10 antihistamine compounds in human plasma using pipette tip solid-phase extraction and gas chromatography/mass spectrometry. An HPLC-based analytical method for the simultaneous screening of antihistamine compounds was reported by Arayne et al. (2011) and Neela et al. (2008). However, most of these methods are limited for assaying antihistamine compounds
in HPLC. There are few methods for the simultaneous analysis of LC–MS/MS in health food products. Also, LC– MS/MS method was specificity, selectivity, and sensitivity enabled rapid analysis. To increase analytes specificity, selectivity, and sensitivity from the sample matrix, a re-verse-phase LC–MS/MS method was developed.
In this study, we attempt to extend previous work by simultaneously monitoring for the adulteration of 22 anti-histamine compounds in dietary supplements. Health foods sold in Korean markets, suspected of being adulterated with 22 illegal antihistamine compounds, were screened simultaneously using LC–MS/MS.
Methods and materials Standards and reagents
The reference standards of the 22 antihistamine com-pounds, namely, acrivastine, astemizole, azelastine hy-drochloride, brompheniramine, clemastine fumarate salt, cetirizine, chlorpheniramine maleate, cyproheptadine, desloratadine, dimenhydrinate, diphenhydramine hy-drochloride, ebastine, epinastine hyhy-drochloride, fexofe-nadine, hydroxyzine hydrochloride, ketotifen fumarate salt, levocetirizine dihydrochloride, loratadine, olopatadine, promethazine, terfenadine, and triprolidine were purchased from Sigma-Aldrich (St. Louis, MO, USA) and USP (Rockville, MD, USA). The molecular formulas and structures of the compounds are shown in Table 1.
Acetonitrile and methanol were purchased from Merck (Darmstadt, Germany). Sodium phosphate and phosphoric acid (HPLC-grade) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Purity grade water was prepared using a Millipore membrane system (Millipore, Bedford, MA, USA). Stock solutions of the analytical standards were prepared separately in methanol at a concentration of 1,000 lg/mL and stored at 277 K.
Samples
Among the 117 samples, including 33 capsules, 32 tablets, 24 liquids, 20 powders, two pills, and six other drugs, suspected of being adulterated with undeclared synthetic drugs, 45 were purchased from online sites, and the remaining 72 were ob-tained from drug shops and markets in South Korea. The 117 samples consisted of 67 anti-obesity drugs, 40 immunity en-hancer drugs, eight anti-allergy drugs, and two other drugs. Sample preparation
Table 1 Chemical structure of 22 antihistamines
Table 1continued
Table 1continued
of the homogenized sample was extracted with 20 mL methanol by sonicating for 10 min and filtering through a 0.22-lm polytetrafluoroethylene (PTFE) filter. Then, the solution was diluted with methanol and subjected to LC– MS/MS analysis.
LC–MS/MS conditions
LC–MS/MS analysis was performed using an ultra-per-formance liquid chromatograph coupled to a Waters Xevo TQ mass spectrometer (Waters, Milford, MA, USA). An electrospray ionization (ESI) source was used in the posi-tive mode, and data were collected using the Masslynx 4.1 software. The analytes were separated on an Acquity UPLC BEH C18 column (100 9 2.1 mm ID, 1.7 lm;
Waters, Milford, MA, USA). Gradient elution was carried out using 0.1 % formic acid in water (eluent A) and 0.1 % formic acid in ACN (eluent B). Over the course of 7 min, the eluent comprising a mixture of 95 % A and 5 % B was gradually changed to 100 % B. After 8 min, gradient was changed to the initial concentration. The injection volume was 2 lL. The sample was introduced into the ESI system at a flow rate of 0.25 mL/min so that optimal LC–MS/MS conditions could be used. In the positive ionization mode, the ESI mass spectrometry parameters were as follows: desolvation temperature, 673 K, capillary voltage 2.7 kV, desolvation gas flow at 600 L/h, and collision gas flow at 0.25 mL/min.
Results
Optimization of sample preparation
Considering the differences in the structure and properties of the various analytes, we tried to optimize the method by varying the following parameters: (i) solvent (ethanol and methanol), (ii) amount of solvent (50, 70, and 100 %), and (iii) sonication time (from 10 to 60 min). The efficiency of the extraction procedure was checked using a recovery test. In the first set of experiments, the extraction efficiencies of methanol and ethanol were compared; good recovery
efficiencies were observed in methanol (95.50–133.14 %) and ethanol (103.10–136.79 %). There was no significant difference in the extraction efficiencies; however, we choose methanol as the extraction solvent because methanol is the most stable of all alcohols (Adams et al.2000). In the second set of experiments, we determined the optimum ratio of solvent to water (50, 70, and 100 %). These experiments were performed only with methanol as it gave the highest recovery rate in the previous experiments. Our results showed that the use of 50 % (28.75–108.42 %) and 70 % (68.17–102.44 %) methanol resulted in lower extraction efficiencies than those obtained with 100 % methanol. In the case of 100 % methanol, a good response and proper peak shapes were obtained for all the analytes, and the mean re-covery rates ranged from 76.96 to 109.14 %. Therefore, 100 % methanol was chosen for the purposes of extraction. In the third set of optimization experiments, we determined the optimal sonication time ranging from 10 to 60 min. Recovery efficiencies of 10, 30, and 60 min sonication were 94.35–108.35, 51.53–113.58, and 53.18–103.58 %, respec-tively. The extraction efficiency at 10 min is slightly higher than the other two. Since one of our aims was to develop a rapid analytical method, a sonication time of 10 min was finally chosen for extraction. From the results of these op-timization studies, we concluded that samples should be extracted with 100 % methanol by sonicating for 10 min (Fig.1). All tests were carried out in triplicate.
Optimization of LC–MS/MS conditions
The obtained multiple reaction monitoring (MRM) transi-tion parameters of LC–MS/MS for the determinatransi-tion of the 22 antihistamine compounds are summarized in Table2. The exact mass of a precursor ion (Q1) was obtained from its respective protonated molecular ion [M ? H]?. MS spectra were studied in both positive and negative ion modes. Compared to the negative ion mode, antihistamine compounds had not only a higher sensitivity but also a clearer mass spectrum in the positive ion mode, making it easier to identify peaks corresponding to molecular ions or quasi-molecular ions. Therefore, the positive MS ion mode was selected for further studies. Antihistamine compounds
Table 1continued
No. Compound name Molecular formula (Molecular weight) Chemical structure 22 Triprolidine C19H22N2(278.39)
N
were easily ionizable in positive mode using an electro-spray ionization source (ESI) and gave a strong protonated molecule [M?H]?. Notably, the adducted form [M?CH3]
of cetirizine exhibited a higher intensity than its protonated molecular ion. The precursor–product ion pairs for MRM detection were generated by the Intellistart (automatic tuning and calibration of the waters Xevo TQ-S) protocol, which was embedded in the MassLynx software; other-wise, the signal of each compound has to be manually optimized by altering the cone voltage and collision ener-gy. Two or three product ions (Q3) of each compound were selected among the fragmented ions from Q1 based on their high intensities and absence of apparent cross contribution or other interferences. The Q3 ion having the highest in-tensity was selected as the ion to be used for quantification. For example, chlorpheniramine, a histamine receptor an-tagonist, which is commonly incorporated individually or in conjunction with decongestants in pharmaceutical compositions, showed an intense peak corresponding to [M?H]?ions at m/z = 275. The most abundant ions found in the product ion mass spectra for chlorpheniramine cor-responded to peaks at m/z = 167.06, 201.00, and 230.05 at collision energies of 35, 34, and 10 eV, respectively. The peak at m/z = 230 was attributed to the ionic fragment obtained after the loss of dimethylamine [NH(CH3)2]. The
major fragment ion at m/z = 167.0 corresponds to the loss of [C4H11ClN] (Fig.2).
Fig. 1 Flow chart of sample preparation steps for antihistamine in food
Table 2 MRM condition for 22 antihistamines
Table 2continued
Limit of detection (LOD) and limit of quantification (LOQ)
The LOD and the LOQ of the 22 illegal antihistamine compounds were determined by spiking the solid (hard capsules, soft capsules, tablets, pills, and powder) and liquid samples with the analytes. The LOD and LOQ were defined as minimum detectable concentrations in a sample matrix at a signal-to-noise (S/N) of three and ten, respec-tively. The lowest LOD of the instrument (0.0003 lg/mL) was observed for clemastine and loratadine, whereas the highest LOD (0.3 lg/mL) was observed for astemizole. The LOQ of the instrument was determined to range from 0.0009 to 0.6 lg/mL. For a 20-mL solution prepared from 1 g of the solid sample, the LOD and LOQ of the method were determined to be in the range of 0.006–6.0 and 0.018–12.0 lg/mL, respectively. The LOD and LOQ of the
The linear range for the quantification of compound levels was obtained by diluting each stock solution with methanol at six different concentrations. In all instances, the corre-lation coefficients were shown to be between 0.997 and 0.999.
Application of the method
All the 117 samples were assayed with this LC–MS/MS method. The results are shown in Table3. In order to evaluate the recovery efficiency, we compared the response of each target compound spiked into a blank solid- or liquid-type sample in triplicate at a concentration of 1.0 lg/mL. The mean recoveries of the compounds were determined to be 89.70–108.50 % (spiked in liquid type) and 93.20–111.80 % (spiked in solid type). As the result, the health food samples investigated, diphenhydramine was
samples. Diphenhydramine can cause side effects such as sedation, sleepiness, dizziness, disturbed coordination, and epigastric distress. In light of this safety management for adulterated, antihistamine of health food in Korea was well carried out relatively. But need for continuous monitoring of health food is steadily increasing, more attention should be paid to the safety of health food in Korea.
Discussion
In the present study, the monitoring of 117 health food products for adulteration by 22 antihistamines was carried out by LC–MS/MS. Procedures for sample preparation and analysis were optimized. The method has been applied for 117 health food, and diphenhydramine was detected one of the health food samples. Selling counterfeit or adulterated food products may cause serious risks to consumers, especially if the goods do not comply with legal regula-tions, thus becoming threats on food safety. Also, it is very dangerous for consumers to take unknown amounts of such adulterated food products and this is becoming a noticeable problem throughout the world. There is a steady increase in the number of cases of antihistamine adulterants in dietary supplements. Therefore, continual research on monitoring
adulterated dietary supplements, food products, and med-icinal products should be pursued for safeguarding human health.
Acknowledgments This research was supported by a Grant (12181MFDS705) from Ministry of Food and Drug Safety in 2013.
References
Adams MC, Yamada Y, Yagi M, Kondo T, Wada T (2000) Stability of methanol, propanol, and SF6 as high-temperature tracers. Proc World Geotherm Congr 2000:3015–3019
Anon (2010) NBJ’s global supplement and nutrition industry report. 2010.http://www.nutritionbusinessjournal.com
Arayne MS, Sultana N, Mirza AZ (2011) Simultaneous determination of gliquidone, pioglitazone hydrochloride, and verapamil in formulation and human serum by RP-HPLC. J Chromatogr Sci 49:114–117
Bogusz MJ, Huda H, AI-Enazi E, Ibrahim Z, Al-Tufail M (2006) Application of LC-ESI-MS-MS for detection of synthetic adulterants in herbal remedies. J Pharm Biomed Anal 41:554–564
Bouldin AS, Smith MC, Graner DD, Szeinbach SL, Frate DA, Croom EM (1999) Pharmacy and herbal medicine in the US. Soc Sci Med 19:279–289
Chan TY, Critchley JA (1996) Usage and adverse effects of Chinese herbal medicines. Hum Exp Toxicol 15:5–12
Table 3 Measured
concentration (lg/kg) ranges of the 22 antihistamine compounds in health food products from a market in South Korea
N.D. not detected
Sample Anti-obesity Anti-allergy Immune enhancer etc.
Chan TY, Chan JC, Tomlinson B, Critchley JA (1993) Herb-induced aconitine poisoning. Lancet 341:370–371
Deconinck E, Verlinde K, Courselle P, Beer JO (2012) A validated ultra high pressure liquid chromatographic method for the characterization of confiscated illegal slimming products con-taining anorexics. J Pharm Biomed Anal 59:38–43
Gergov M, Robson JN, Ojapera I, Heinonen OP, Vuori E (2001) Simultaneous screening and quantitation of 18 antihistamine drugs in blood by liquid chromatography ion spray tandem mass spectrometry. Forensic Sci Int 121:108–115
Hasegawa C, Kumazawa T, Lee XP, Fujishiro M, Kuriki A, Marumo A, Seno H, Sato K (2006) Simultaneous determination of ten antihistamine drugs in human plasma using pipette tip solid phase extraction and gas chromatography/mass spectrometry. Rapid Commun Mass Spectrom 20:537–543
Hel KO, Lee EH, Kim SW, Kim KJ, Cha KO (2006) Consumer’s trend and market. Kyomunsa, Seoul, pp 79–89
Korea Food and Drug Administration (2012) A study for improving food & drug statistical year book. Korea Food and Drug Administration, Korea
Ku YR, Wen KC, Ho LK, Chang YS (1999) Solid-phase extraction and high performance liquid chromatographic determination of steroids adulterated in traditional Chinese medicines. J Food Drug Anal 7:123–130
Lee HM, Kim CS, Jang YM, Kwon SW, Lee BJ (2011) Separation and structural elucidation of a novel analogue of vardenafil included as an adulterant in a dietary supplement by liquid chromatography-electrospray ionization mass spectrometry, in-frared spectroscopy and nuclear magnetic resonance spec-troscopy. J Pharm Biomed Anal 54:491–496
Miller GM, Stripp R (2007) A study of western pharmaceuticals contained within samples of Chinese herbal/patent medicines collected from New York City’s Chinatown. Leg Med 9:258–264
Neela MB, Ganbavale SK, Bhatia MS, More HN, Kokil SU (2008) RP-HPLC and spectrophotometric estimation of ambroxol hydrochloride and cetirizine hydrochloride in combined dosage form. Indian J Pharm Sci 70:603–608
Rebiere H, Guinot P, Cirade C, Bonnet PA, Nicolas A (2012) Detection of hazardous weight-loss substances in adulterated slimming formulations using ultra-high-pressure liquid chro-matography with diode-array-detection. Food Addit Contam Part A 29:161–171
