1. Introduction

Diabetes is a chronic disease characterized by highblood sugar levels. It is classified into three major types: Type one (I), type two (II), and gestational diabetes. Type II diabetes accounts for 90 % of the cases,¹ and oral antidiabetic drugs constitute the main therapy for most patients required to controltheir blood glucose.² Because of concerns over some of the potential side effects of these drugs, e.g., obesity, weakened immunity, aggravated inflammation, and death caused by hypoglycemic shock from themisuse of medication, dietary supplement and herbal medicine sales have increased as they are considered safer than medication (e.g., synthetic drugs) for treatinghealth in an “all-natural” manner.^3-6 Nevertheless, somemanufacturers add illegal adulterants to their products to achieve drastic effects in a short time period. Several unlabeled illegal adulterants have beendetected in dietary supplements, which can potentially cause serious risks to public health.⁷

Several antidiabetics have been reportedly detected. In Saudi Arabia, 7.5 mg of glibenclamide has beendetected from tablets and 4.5 mg from powders.⁸ In China, metformin, glimepiride, and phenforminhave been detected in counterfeit dietary and herbal supplements. ⁹ Furthermore, in Singapore,¹⁰ an outbreak of severe hypoglycemia has been reported, caused by the contamination of illegal sexual enhancement drugs with glyburide. As much as 13-100 mg of gly buride has been detected in blood and urine from 127 non-diabetic patients (among 127, 4 people died). In 2008, similar cases of glibenclamide-induced hypoglycemia have been reported in Hong Kong. Other illegal sexual enhancement brands were implicated in these studies.¹¹ Hence, it is imperative to develop a simultaneous method to comprehensivelyscreen rapidly. The most widely applied methodologies for pharmaceutical analysis include high-performanceliquid chromatography (HPLC),^12-18 high-performanceliquid chromatography-tandem mass spectrometry (LC-MS/MS), and spectroscopic methods.^19-23 Almost the whole studies have reported the analysis for thescreening of adulterants in urine, blood, body fluids, and plasma. Some of studies described such amethod that procedure for screening, identification, and quantification of several antidiabetic drugs in foods and herbal products by LC-MS/MS^8-11 Using LC-MS/MS, typically a particular peak from the mass spectrum is selected and isolated and collisions are induced within the mass spectrometer to force acharacteristic fragmentation of the selected ion. The LC-MS/MS can detect slight amount than UPLC and further increase the specificity.

This paper proposed an LC-MS/MS method for the rapid, reliable detection of 26 antidiabeticadulterants in dietary supplements. Multiple reaction monitoring (MRM) was employed to monitor the LC effluent by simultaneously using selected transitions for each compound, where reliability can be improved based on the fact that the relative peak areas maintaingood stability. To the best of our knowledge, such a procedure, involving the analysis of such a comprehensive list of compounds, has not been reported thus far. Therefore, there is a clear requirement for comprehensive screening of illegal adulterants 26 antidiabetic adulterants in dietary supplement.

2. Materials and Methods

2.1. Chemicals and reagents

Reference standards of sulfonylureas or meglitinides (carbutamide, chlorpropamide, glibenclamide, glibor-nuride, gliclazide, glimepiride, glipizide, gliquidone, glymidine, tolazamide, tolbutamide, mitiglinide, nateglinide, repaglinide, alogliptin benzoate), biguanidesor thiazolidinedianes (buformin, metformin, phenformin, pioglitazone, rosiglitazone, troglitazone), Dipeptidyl Peptidase (DPP)-4 inhibitors (sitagliptin and vild agliptin), and SGLT(The sodium/glucose cotransporter) 2 inhibitors (canagliflozin, empagliflozin, ipragliflozin) were purchased from USP (Rockville, MD, USA), Sigma-Aldrich (St. Louis, MO, USA), Toronto Research Chemicals (Toronto, ON, Canada), and Santa Cruz (Dallas, TX, USA). HPLC-grade methanol was purchased from Burdick and Jackson (Muskegon, MI, USA), and formic acid was purchased from Sigma-Aldrich (St. Louis, MO, USA). Milli-Q water(18.1 mΩ) from a Milli-Q purification system (Millipore, Bedford, MA, USA) was used throughout.

2.2. Sample preparation

Stock solutions were prepared by dissolving standards in methanol at approximately 1 mg mL⁻¹ and stored in a refrigerator (2-8 °C) until use.

Dietary supplements were pulverized into powders starting from various samples (i.e., tablets, hard capsules, soft capsules, powders, liquids, and pills), and 1 g of the powder sample was dissolved in 70 % methanol. Capsule and soft gel shells were excluded. After 30 min of sonication for complete dissolution, the sample solution was added into a 50 mL volumetric flask, and its volume was made up to the mark. The sample was then filtered through a 0.22 µm polytetrafluoroethylene (PTFE) filter (Millipore, Milford, USA) before analysis.

2.3. Instrumentation

Analytes were separated on an Agilent 1200 series HPLC instrument (Agilent Technologies, Palo Alto, CA, USA). The LC system consisted of a quaternary pump, a vacuum degasser, and an autosampler. Data in the positive and negative modes were obtained by anelectrospray ionization (ESI) source via an API 4000 triple quadrupole mass spectrometer (AB Sciex, Concord, ON, Canada).

2.4. Analytical method

2.4.1. LC-MS/MS operating conditions

Compounds were separated by a Shiseido Capcell pak ® C18 MG-II column (2.0 mm × 100 mm, 3 µm particle size) to improve the peak shape and MS detection sensitivity of the target compounds. A lineargradient system of mobile phase A (0.1 % formic acid indistilled water) and mobile phase B (0.1 % formic acid in MeOH) was used at a flow rate of 0.3 mL/min. Table 1 summarizes the gradient program and MS/MS conditions.

Table 1. The condition of LC-MS/MS

3. Method Validation

The following method parameters were evaluated to validate the reliability of the proposed method: specificity, linearity, limit of detection (LOD), limitof quantitation (LOQ), precision, accuracy, recovery, and stability. The range of linearity was tested by analyzing six standard calibration solutions in triplicateat concentrations set at 1, 2, 4, 8, 10, and 20 times of the LOQ. According to the recommended guidelines, LODs and LOQs were determined by spiked samples based on the signal-to-noise ratios of 3:1 and 10:1, respectively. The accuracy of intra- and inter-dayexperiments were evaluated in triplicate at low (near the LOQ), medium (~5-fold above the LOQ), and high(~10-fold above the LOQ) concentrations to a blanksample containing a dietary supplement. Intra- andinter-day precision was evaluated three times by analysison the same day and on three days, and the relativestandard deviation (% RSD) values were determined foreach compound. Recovery was determined as the standard area as compared to the blank sample areaspiked with 26 antidiabetic compounds. The blanksample was analyzed along with six types of samples (powders, pills, hard and soft capsules, liquid, and tablets, respectively) containing three concentrations of the mixed standard solutions. The stability of the 26 antidiabetic compounds solution was evaluated by quantitative determination at several time points over 48 h. The stability of 26 compounds was assessed by processing solutions after 6 h storage at room temperature. Then, the autosampler stability was determined by keeping the reconstituted samples forapproximately 24 and 48 h in an autosampler at 4 ℃ before analysis. Method validation was performed according to the requirements published by the ICH guidelines. ²⁷

4. Method Application

Seventy-eight samples collected from Korean online or offline markets and eight samples requested from the criminal investigation public office were used, including those from tablets (9), hard capsules (10), soft capsules (12), powders (14), liquids (26), and pills (15).

5. Results and Discussion

5.1. Optimization of mass spectrometric and chromatographic conditions

All diabetic compounds were evaluated using the LC-MS/MS method. Standard solutions containing 20-100 ng/mL in methanol were infused directly into the ESI source of the mass spectrometer. Prominent protonated molecular ions [M+H]⁺ and [M+NH₄]⁺ in the positive mode, as well as prominent protonated molecular ions [M-H]− in the negative mode, were observed in the full scan mass spectra. Ammoniumadducts ion [M+NH₄]⁺ in positive mode was adopted for Canagliflozin and ipragliflozin identification. In previous reported studies, ammonium adducts ions for canagliflozin and ipragliflozin in the presence offormic acid were more predominate and sensitive then hydrogen adduct ions [M+H]⁺.^24-26 Collisionenergies were optimized for each analyte to obtain the most intense fragment ions. Multiple reaction monitoring (MRM) transitions were initially monitored for each analyte. During the collision of the precursorions in tandem MS, more than two daughter ions of the 26 antidiabetic compounds were obtained, highest peak for quantitation and another for confirmation purpose (Fig. 1, Table 2). To optimize peak shape with appreciable retention times, various columns were investigated. Three columns, HILIC, C18, and C8, were used. The best resolution and intensity were observed with the Shiseido Capcell pak® C18MG-II column (2.0 mm × 100 mm, 3 μm particlesize), without excessive tailing in 18 min. In addition, the separation of these antidiabetic compounds was attempted using various combinations of acetonitrile, methanol, and water with different percentages of buffers. The best separation was achieved using 0.1% formic acid with methanol and water. Because of the high selectivity and efficiency of LC-MS/MS, the simultaneous separation of 26 antidiabeticcompounds has been reported for the first time, to the best of our knowledge.

Fig. 1. Individual TIC of 26 anti-diabetic compounds.

Table 2. MRM conditions for 26 anti-diabetic compounds

5.2. Method validation

Specificity was confirmed by the MRM transition parameters using the precursor and product ion pairs of UPLC-MS/MS for the determination of the 26 compounds summarized in Fig. 2 and 3. The blankand spiked samples of selected ions from the mass spectra obtained using LC/MS/MS, no interfering peaks were observed at the retention times. Under the optimized conditions, a linear relationship with agood correlation coefficient (R2 > 0.99, n = 3) was observed between the peak area ratios and the concentrations of 26 compounds in the range of 0.5~1200 ng/mL. The limits of detection and quantitation (LOD/LOQ) were mearsured in solid sample (LOD, 0.16~20.00 ng/mL; LOQ, 0.50~60.00 ng/mL) and in liquid sample (LOD, 0.16~20.00 ng/mL; LOQ, 0.50~60.00 ng/mL), respectively (Table 3). The accuracy values from the intra-day analysis was 81.99~112.64 %, whereas the values for the inter day analysis were 80.69~118.81 %. The RSD of precision was ≤ 10.25 % in intra-day and ≤ 13.19 % in inter-day (Table 4). Extraction recovery was also investigated by analyzing one of the commercial products that had been spiked with the standard analytes. The mean overall recoveries (with the precision) of all the analytes were summarized in Table 5. The stability of 26 antidiabetic compounds up to 48 hr was less than 14.88 % (Table 6). The results showed that the method met the desired level of acceptance criteria and hence was considered accurate and precise for the analysis of 26 antidiabetic adulterants in dietary supplement and other herbal products in the area of forensic science.

Fig. 2. TIC of (a) blank (b) anti-diabetic compounds in positive mode

Fig. 3. TIC of (a) blank (b) anti-diabetic compounds in negative mode.

Table 3. The linearity, LOD and LOQ of 26 anti-diabetic compounds

Table 5. Extraction recovery of 26 anti diabetic compounds in dietary supplement samples (six types) by LC-MS/MS

Table 6. Stability of anti-diabetic over 24 h, as determined by LC-MS/MS

5.3. Method application

The established UPLC-MS/MS method wassuccessfully applied to the determination of 26 antidiabetic compounds in six dietary supplementsamples, used to prevent diabetics, collected from acriminal investigation public office and online or of fline Korean markets. Sample preparation was carried out as described in Sample Preparation in the Materials and Methods section. Of the 26 antidiabeticcompounds, glibenclamide and pioglitazone weresimultaneously detected in one capsule and confirmed (Fig. 4) at concentrations of 1.52 and 0.53 mg (percapsule), respectively. Based on this result, the proposed method is rapid, reliable, and accurate, with good applicability.

Fig. 4. Mass spectra of a standards and sample that contains the pioglitazone and glibenclamid. (a) pioglitazone STD, (b) pioglitazone in sample, (c) glibenclamid STD, (d) glibenclamid in sample

6. Conclusions

In this study, an accurate, reproducible method based on liquid chromatography coupled withelectrospray ionization tandem mass spectrometry was developed and validated for the simultaneous determination of 26 antidiabetic compounds in dietary supplements. In addition, this study exerts significantadvantages over earlier reported methods, e.g., simultaneous quantification of 26 antidiabetic analytes, shorter run time, wider linearity range with high sensitivity, and simple reproducible extraction. The developed method was successfully validated. Using the validated method, glibenclamide as an insulinsecretagogues and pioglitazone as an insulin actionenhancer were simultaneously detected from a capsulardietary supplement. These medicine compounds can be dangerous for consumers without a doctor ' sprescription. From the results of validation parameters, the developed simultaneous analysis method can be used for the routine analysis of antidiabetic compoundsin various forms of dietary supplements. This advanced analytical method might help to restrict the use ofillegal adulterants in dietary supplements.

Acknowledgements

This study was supported by a grant (Number: 15181MFDS521) from the Ministry of Food and Drug Safety in 2016.