Histamine, a low-molecular-weight amine, is a natural body constituent. Histamine is synthesized from l-histidine by histidine decarboxylase and involved in neurotransmission, allergic inflammation, and immune modulation. Histamine exerts its effects through the interaction with one of the four distinct receptors (H1, H2, H3, and H4). The manifestation of receptors is changed depending on the level of cell differentiation and microenvironmental influences.1 Histamine exhibits proinflammatory activity and is relevant to the development of diverse aspects of antigen-specific immune response by the H1 receptor.2-3,5 H1 antihistamines are used in the treatment of allergic disorder, allergic rhinoconjunctivitis, and urticaria.4,6 H1 antihistamines directly inhibit the action of histamine through H1 receptors on antiallergic inflammation in blood vessels and nerve cells. Moreover, antiallergic activities such as the anti-inflammatory functions of mast cells and basophils probably exert a direct inhibitory effect on calcium ion channel and NF-kB.5,7 H1 antihistamines are classified into alkylamines, piperazines, piperidines, ethanolamines, ethylenediamines, and phenothiazines according to the chemical structures or functional groups, which is determined by the permeability of the blood–brain barrier for classification as the first-or second-generation histamines.5 There are large differences between the two H1 antihistamine generations in terms of their propensity to cause the side effects of central nervous system (CNS). The first-generation H1 antihistamines penetrate well into the CNS where they induce sedation. In contrast, the second-generation H1 antihistamines allow much reduced CNS sedation.6,8 According to the United States Food and Drug Administration-approved product labelling, potential side effects associated with first-generation antihistamines include cardiotoxicity, drowsiness, and cognitive impairment.24
Because of the growing interest in health and nutrition, the sale of dietary supplements steadily increases every year. Unfortunately, the adulteration of food ingredients and dietary supplements with various types of drugs is a concern, risking human health in long term. Liang et al. 23 reported that promethazine as adulterant was found in herbal medicine and food supplements, these products were declared having the effect of tranquilization or improving ‘health status’ of old people. The consumption of these adulterated products which contain chemical drugs could be harmful to consumers with various health risks. For this reason, more accurate and effective method is needed for rapid determination of adulteration such as antihistamines in foods or dietary supplements.
Previously, the simultaneous analysis of antihistamines using various instruments has been reported. HPLC has been used to analyze antihistamines by Arayne et al. 9 and Karakus et al. 10 Gergov et al. 11 simultaneously determined the concentration of 18 antihistamines in blood by liquid chromatographyionspray tandem mass spectrometry (LC–MS/MS) with multiple reaction monitoring (MRM). Hasegawa et al. 12 simultaneously determined the concentration of antihistamines such as diphenhydramine, chlorpheniramine, triprolidine, promethazine, cyproheptadine, and clemastine by gas chromatography coupled with mass spectrometry (GC–MS). Moreover, levocetirizine and its isomers in human blood have been identified by LC–MS/MS. Thus, most of the simultaneous determinations of the concentrations of antihistamines have been conducted only in the blood or urine samples from human, not in adulterated food or dietary supplements, and by liquid chromatography coupled with UV detection (LC–UV) or LC–MS.13-21
In this paper, we report a rapid and accurate method for the simultaneous determination of the concentrations of antihistamines in various types of dietary supplements that were intentionally adulterated with antihistamines by LC–MS/MS.
2. Materials and Methods
2.1. Reagents and chemicals
Acrivastine (ACV), astemizole (AMZ), azelastine hydrochloride (AZT), brompheniramine maleate (BPA), cetirizine (CTZ), chlorpheniramine maleate (CPA), clemastine fumarate salt (CMT), cyproheptadine hydrochloride sesquihydrate (CHD), desloratadine (DLD), diphenhydramine hydrochloride (DPH), ebastine (EBT), epinastine hydrochloride (EPT), fexofenadine hydrochloride (FFD), hydroxyzine hydrochloride (HXZ), ketotifen fumarate salt (KTF), loratadine (LTD), olopatadine hydrochloride (OPD), promethazine hydrochloride (PMZ), terfenadine (TFD), and triprolidine hydrochloride (TPD) were purchased from USP (Rockville, MD, USA) and Sigma-Aldrich (St. Louis, MO, USA). HPLC-grade methanol and acetonitrile were purchased from Burdick & Jackson (Muskegon, MI, USA). Formic acid was purchased from Sigma-Aldrich (St. Louis, MO, USA). High-purity deionized water (DW) was prepared using Milli-Q (Millipore, Bedford, MA, USA) purification system. The standard stock solutions (1,000 µg/mL) were prepared by dissolving an appropriate amount of the drug in methanol. Each stock solution was stored at 4 ℃. Standard working mixtures were prepared daily by diluting the stock solutions with methanol for each calibration curve and method validation.
A Waters Acquity UPLC system (Waters, Milford, MA, USA) equipped with binary pumps, a sample manager, and a column oven was used for the experiments. The liquid chromatographic separation was performed using an Acquity UPLC BEH C18 (1.7 µm, 2.1 × 100 mm, Waters, Milford, MA, USA) column with the temperature of the column set at 40 ℃. The flow rate was 0.25 mL/min, and the injection volume was 2 µL. The mobile phase consisted of 0.1% formic acid in DW (A) and 0.1% formic acid in acetonitrile (B). The gradient condition of the mobile phase was as follows: 0 min, 5% B; 1.0 min, 5% B; 7.0 min, 100% B; 8.0 min, 100% B; 8.1 min, 5% B; and 10.0 min, 5% B. An MS system manufactured by Waters Xevo TQMS (Waters, Milford, MA, USA) was operated using MRM with electrospray ionization (ESI) in positive ion mode. The desolvation temperature was 400 ℃, and the flow rates of the desolvation and cone gases were set at 600 L/h and 40 V, respectively. For optimizing the analytical conditions, the MS capillary voltage was set at 2.7 kV.
2.3. Sample preparation
We purchased different types of dietary supplements such as powders, capsules, tablets, liquids, and pills from the South Korean markets for method validation. Each sample was homogenized, and 1 g of the sample was placed in a 20 mL volumetric flask. Then, the sample was extracted with methanol by sonication for 10 min. Finally, the extract was filtered through a 0.22-µm PVDF filter (Millipore, Milford, MA, USA) before the injection for analysis.
3.1. Optimization of LC-MS/MS conditions
The MS/MS optimization was performed by the direct infusion of each individual standard solution. Precursor ion discovery (PID) and LC-MS/MS scans were carried out using ESI operated in positive mode. The protonated molecular ion and two or three fragment ions were selected as the precursor and product ions, respectively. The instrument parameters such as collision energy, cone voltage, desolvation temperature, and capillary voltage were tuned to obtain the optimal intensity of product ion. The optimized MRM conditions are shown in Table 1. To optimize the separation of 20 antihistamine compounds, various compositions of mobile phase with different buffers such as ammonium acetate and formic acid were used.
Table 1.a)Multiple monitoring reaction
The mobile phase without any buffer showed low peak intensity. Moreover, a broad peak shape and tailing were observed when ammonium acetate buffer was used, while the use of formic acid showed a good peak intensity and separation. When an Acquity UPLC BEH C18 column was used, the compounds could be effectively separated in terms of peak shape and intensity compared to those obtained using BEH C8 (1.7 µm, 2.1 × 100 mm, Waters, Milford, MA, USA) and HSS T3 column (1.8 µm, 2.1 × 100 mm, Waters, Milford, MA, USA). Therefore, the BEH C18 column was selected because it showed the best separation of the 20 antihistamine compounds. The LC–MS/MS chromatograms of the 20 antihistamine compounds are shown in Fig. 1.
Fig. 1.Typical chromatogram of LC–MS/MS for 20 antihistamines: 1, fexofenadine (FFD); 2, terfenadine (TFD); 3, ebastine (EBT); 4, astemizole (AMZ); 5, cetirizine (CTZ); 6, loratadine (LTD); 7, azelastine (AZT); 8, hydroxyzine (HXZ); 9, acrivastine (ACV); 10, clemastine (CMT); 11, olopatadine (OPD); 12, brompheniramine (BPA); 13, desloratadine (DLD); 14, ketotifen (KTF); 15, cyproheptadine (CHD); 16, promethazine (PMZ); 17, triprolidine (TPD); 18, chlorpheniramine (CPA); 19, diphenhydramine (DPH); 20, epinastine (EPT).
3.2. Method validation
The developed LC–MS/MS method was validated according to the International Conference on Harmonization (ICH) guidelines.22 The method was validated with regard to specificity, limit of detection (LOD), limit of quantification (LOQ), linearity, precision, accuracy, recovery and stability.
The specificity of the method was evaluated by comparing the chromatograms obtained from the blank sample and spiked blank sample. The analysis of the 20 antihistamine compounds was carried out by injecting a blank sample (a pill type), and then the blank sample was spiked with the antihistamine compounds. The LC–MS/MS analysis of the 20 antihistamine compounds showed good sensitivity without interference from the sample matrix. Fig. 2 shows the chromatograms of the blank and spiked samples. The other forms of dietary supplements showed a similar trend, such as pills (data not shown).
Fig. 2.MRM chromatograms of LC–MS/MS for 20 antihistamines: (a) blank sample, (b) spiked sample with antihistamines: Peak assignments: 1, fexofenadine (FFD); 2, terfenadine (TFD); 3, ebastine (EBT); 4, astemizole (AMZ); 5, cetirizine (CTZ); 6, loratadine (LTD); 7, azelastine (AZT); 8, hydroxyzine (HXZ); 9, acrivastine (ACV); 10, clemastine (CMT); 11, olopatadine (OPD); 12, brompheniramine (BPA); 13, desloratadine (DLD); 14, ketotifen (KTF); 15, cyproheptadine (CHD); 16, promethazine (PMZ); 17, triprolidine (TPD); 18, chlorpheniramine (CPA); 19, diphenhydramine (DPH); 20, epinastine (EPT).
3.2.2. Limit of detection (LOD) and limit of quantification (LOQ) and linearity
The LOD and LOQ were determined from the standard deviation of the response of the injected target compounds prepared with a spiked blank sample or reference material (a pill type) at a signal-to-noise ratio of 3 and 10, respectively. The linearity of the compounds was in the concentration range 0.01-20 µg/mL. The determination of the LOD and LOQ were based on a signal-to-noise (S/N) ratio of 3:1 and 10:1, respectively. The LOD and LOQ of the instrument used represent the LOD and LOQ concentrations of the compounds in the analyzed solutions, whereas the LOD and LOQ of the method represent the LOD/LOQ concentrations in the dietary supplements investigated. The LOD and LOQ of the instrument were determined as 0.0003-0.3 and 0.0009–0.6 µg/mL, respectively. The calibration curves of the 20 antihistamine compounds were constructed at six different concentration levels, and they showed excellent linear correlation (>0.99) (Table 2).
Table 2.Summary of the calibration curve, limit of detection (LOD), limit of quantification (LOQ)
3.2.3. Accuracy and precision
The intra-day and inter-day assay precision were determined by repeated analysis in a single day (n = 3) and three different days (n = 3), respectively, and evaluated by the relative standard deviation (RSD). The intra-day and inter-day assay accuracy of the method was determined by adding a known amount of each compound corresponding to six independent concentration levels in triplicate. The intra-day and inter-day accuracy of the method calculated as the recovery. The RSD of the recovery was 0.3-9.6% (inter-day precision) and 0.1-9.8% (intra-day precision) for all the antihistamines (Table 3). The accuracy of the method was calculated by comparing the measured concentrations with the known spiked concentrations in triplicate. As shown in Table 4, the mean of recovery was 82.7-115.0% (intra-day accuracy) and 84.3-113.0% (inter-day accuracy).
Table 3.a)RSD is defined as the standard deviation of a group of values divided by their mean
Table 4.a)RSD is defined as the standard deviation of a group of values divided by their mean
The recovery of method was determined by adding a known amount of target compounds using five replicates at three concentration levels (a low-concentration sample at approximately the LOQ level, a mediumconcentration sample at ~10 times the LOQ level, and a high-concentration sample at ~50 times the LOQ level) in the dietary supplement samples. To evaluate the recovery of antihistamines, the peak areas of the pure standards and spiked samples were compared in five replicates. The mean of recovery of the 20 targeted compounds ranged from 90.5% to 113.7% for tablets, 88.5% to 109.7% for liquids, 88.5 to 116.1 for pills, 84.8% to 117.9% for powders, and 88.0% to 114.6% for capsules. The results are shown in Table 5.
Table 5.a)RSD is defined as the standard deviation of a group of values divided by their mean
The stability of method was evaluated by measuring freshly prepared standard solution of known amount for different period of time. Standard solution of target compounds was measured at several time points up to 24 h. The RSD values of the RSD values were less than 5.9% (Table 6).
Table 6.a)RSD is defined as the standard deviation of a group of values divided by their mean
Previously, most of the simultaneous determinations of the antihistamines have been carried out only in the blood or urine samples from human; these methods can determine food or dietary supplements.
In this method, the chromatogram produced by LC-MS/MS, in addition MS/MS optimization was performed by the direct infusion and LC-MS/MS scans were performed using ESI operated in positive mode. The HPLC separation was achieved on a C18 reverse-phase column by using mobile phase of 0.1% formic acid in DW and 0.1% formic acid in acetonitrile of a gradient elution mode. The optimized method was validated for specificity, linearity, LOD, LOQ, precision, accuracy, recovery and stability according to ICH guideline. The developed method was successfully applied to determine antihistamine compound in dietary supplements without any interference. The results demonstrated that the values were within the acceptable range.
A rapid, selective, and sensitive LC–MS/MS method was developed in this study to analyze 20 antihistamines intentionally adulterated in the dietary supplements. The analytical conditions of the LC–MS/MS method were optimized. The developed method was completely validated and found to perform satisfactorily for the qualitative and quantitative analyses of antihistamines. The method was applied for the analysis of dietary supplements to determine the concentration of illegal drugs.