738 Biomed Environ Sci, 2015; 28(10): 738-750 Original Article Application of UPLC-MS/MS Method for Analyzing B-vitamins in Human Milk* REN Xiang Nan1, YIN Shi An1, YANG Zhen Yu1, YANG Xiao Guang1,#, SHAO Bing2,#, REN Yi Ping3, and ZHANG Jing2 1. National Institute for Nutrition and Health, Chinese Center for Disease Control and Prevention, Beijing 100050, China; 2. Beijing Key Laboratory of Diagnostic and Traceability Technologies for Food Poisoning, Beijing Research Center for Preventive Medicine, Beijing 100013, China; 3. Zhejiang Provincial Centre for Disease Prevention and Control, Hangzhou 310051, Zhejiang, China Abstract Objective To determine ten B-vitamins in human milk by ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Methods The pretreated human milk samples were adequately separated and quantified within 11 min by UPLC-MS/MS with an Acquity UPLC HSS T3 column (2.1×100 mm, 1.8 µm). The mobile phase was a gradient of 2.5 mmol/L ammonium formate aqueous solution and acetonitrile at a flow rate of 0.35 mL/min. Stable isotope internal standards were used in the analysis, to correct for the method variability, including matrix and ionization effects. The homogenized human milk samples were deproteinzed using methanol, unknown contaminants were extracted with diethyl ether and hydrophobic phase was discarded. The analytes were monitored via ESI+ionization and detected in multiple reaction monitoring (MRM) with three acquisition functions. Results Calibration curves ranged from 0.5-160 ng/mL (thiamin, riboflavin, biotin, nicotinic acid, pyridoxine, pyridoxamine, pyridoxal), and 2.5-800 ng/mL (pantothenic acid, FAD and nicotinamide) (R2=0.990-0.999). The relative recovery ranged from 80.1% to 120.2%; accuracy was determined to be 98.3% to 108.0%. Intra-day and inter-day variation were 3.4%-19.9% and 5.9%-18.1%, respectively. The limit of quantification (LOQ) for all vitamins was between 0.25 and 3 µg/L. Conclusion This method was successfully applied for simultaneous analysis of ten B-vitamins in human milk. Key words: B-vitamins; Human milk; UPLC-MS/MS Biomed Environ Sci, 2015; 28(10): 738-750 doi: 10.3967/bes2015.104 ISSN: 0895-3988 www.besjournal.com (full text) CN: 11-2816/Q Copyright ©2015 by China CDC INTRODUCTION meet all nutritional requirements for exclusively breastfed infants aged 0 to 6 months of life[1-3]. It is -vitamins are essential nutrients for important to analyze B-vitamins in human milk growth and development of infants, most because inadequate vitamin intake could lead to of which participate in the metabolism in infantile vitamin deficiencies[4-8]. B [1] the form of coenzyme . Human milk is thought to Adequate intakes (AI) of nutrients, including *This study was supported by the National High Technology Research and Development Program of China (863 Program) (No. 2010AA023004). #Correspondence should be addressed to YANG Xiao Guang, Professor, Tel: 86-10-83132798, Fax: 86-10-83132808, E-mail: [email protected]; SHAO Bing, Professor, Tel: 13381081679; E-mail: [email protected] Biographical note of the first author: REN Xiang Nan, female, born in 1984, PhD, majoring in food nutrition. B-vitamins in human milk by UPLC-MS/MS 739 B-vitamins, are generally estimated by the content in phenomenon of cross-talk may occur if the former human milk from well-nourished mothers and an method is used for analyzing ten B-vitamins, average daily breast milk intake[3,9-10]. The previous which leads to greater deviation of results[29-31]. methods of analyzing B-vitamins, e.g. microbiological, Therefore, it is essential to monitor two or more chemical, radioactive methods, enzyme channels for MRM analysis and optimize the mobile immunoassays, and more recently high performance phase to better separate components to avoid liquid chromatography (HPLC) were usually used for cross-talk[29-34]. the analysis of B-vitamins in human milk[9,11-16]. In To our knowledge, no analytical method has general, each method could be used for only one been published to quantify simultaneously ten individual vitamin with labor-intensive and B-vitamins in human milk. Therefore, we have time-consuming pretreatments[10]. Moreover, a developed a rapid, sensitive, efficient UPLC-MS/MS larger volume of human milk was required for these method for simultaneous analysis of ten B-vitamins determinations[9,11,14-18]. Because of the complexity in human milk. Moreover, this method has been of B-vitamins, constraints of traditional methods and successfully applied to the quantification of thiamin, difficulty of operability of sampling, research studies riboflavin, pyridoxal, pyridoxine, pyridoxamine, of B-vitamins in human milk have been studied in nicotinamide, nicotinic acid, flavin adenine various countries with a limited sample size ranging dinucleotide (FAD), biotin, and pantothenic acid in from 5 to 152 samples[9-16]. human milk. More recently, only Hampel et al.[10] described the analysis of five B-vitamins (thiamin, riboflavin, MATERIALS AND METHODS flavin adenine dinucleotide (FAD), nicotinamide, and [19] pyridoxal) and Tao et al. in China analyzed five Chemicals and Reagents B-vitamins (thiamin, riboflavin, nicotinamide, pantothenic acid, and pyridoxal) in human milk by Thiamin hydrochloride (99% purity), biotin (99% ultra-high performance liquid chromatography- purity), pantothenic acid (99.9% purity), nicotinic tandem mass spectrometry (UPLC-MS/MS). However, acid (98% purity), riboflavin (98% purity), flavin they were not suitable for simultaneously analyzing adenine dinucleotide (FAD) (95% purity), ten B-vitamins, which are necessary in order to nicotinamide (99.5% purity), thiamin- 13 13 establish the data base of human milk and direct the (4-methyl- C-thiazol-5yl- C3) hydrochloride (99 13 13 diet for lactating women. Even though pyridoxal is atom % C, 98% CP), riboflavin- dioxopyrimidine- C4, 15N (99 atom %13C, 98 atom% 15N, 97% CP), the principal form of vitamin B6, pyridoxine and 2 pyridoxamine exist in human milk and the pyridoxal-(methyl-d3) (98 atom % D, 98% CP) were percentages are 2%-16%, 6%-13%, obtained from Sigma-Aldrich Trading Co., Ltd respectively[11,15,20]. Pyridoxine has been used to (Shanghai, China). Pyridoxine hydrochloride (98% treat neonates with early onset seizures. It can purity) and pyridoxamine dihydrochloride (98% improve infants’ intelligence and prevent the purity) were purchased from TCI (Tokyo, Japan). neurologic function damaged[21-23]. The different Pyridoxal hydrochloride (99% purity) was obtained existed forms of each vitamin can be transformed. from Acros Organics (Morris Plains, NJ, USA). Except nicotinamide, nicotinic acid is found in human Acetonitrile and methanol were bought from Dikma milk[9]. Pantothenic acid mostly exists in its free form (HPLC buffer, Beijing, China) and ammonium formate (85%-90%) and bound form (10%-15%)[24]. Pantothenic was obtained from Fluka (Shanghai, China). acid is the universal precursor for coenzyme A (CoA) Ultrapure water was prepared with a Milli-Q and acyl carrier protein and it is closely associated Ultrapure water system (Millipore, MA, USA). with infants’ key metabolic and energy-yielding The human study was approved by the ethics pathways[25-26]. Biotin is found in human milk and can committee of the National Institute of Nutrition and enhance the appetite and prevent the neonatal Food Safety, China CDC. Written informed consent anemia[2,27-28]. These forms are the constituents of was obtained from all participants. Human milk B-vitamins and also contribute to the vitamin samples, included colostrums, transitional milk and functions significantly, but were less studied[10]. mature milk, were collected from apparently healthy Some different components have the same women in Huangpu city in the Guangdong Province quantification ion, e.g. nicotinamide and nicotinic of China and stored at -80 °C freezer in darkness acid, pyridoxamine and pyridoxine. So the until analysis. B-vitamins in human milk by UPLC-MS/MS 741 confirmed by directly injecting the standards (1.0 remove the hydrophobic molecules. The hydrophilic μg/mL) into the mass spectrometer. The optimum phase was transferred to centrifuge tubes and was conditions were as follows: capillary voltage, 3.5 kV; centrifuged at 14,480 xg for 10 min at 4 °C. Finally, source temperature, 120 °C; desolvation 90 µL of the clear supernatant was transferred into temperature, 350 °C; desolvation gas (nitrogen, 99% 150 µL glass insert in a 1.5 mL amber glass vial and purity) flow, 682 L/h; collision gas (argon, 99.99% analyzed. -3 purity) pressure, 4.04×10 mbar. Detection was Both working solutions and samples were acquired in multiple reaction monitoring (MRM) at prepared at the same day and all samples were three functions (time segments). Cone voltage, analyzed within 24 h to ensure the stability of the collision energies and dwell times were optimized analytes. for each compound to get the highest sensitivity and In order to monitor stability of the system accuracy (Table 1). during the process of run, the standard curve, quality control sample in triplicate and pooled milk Quality Control sample were repeatedly injected every 25 injections 0.1 g of NIST SRM 1849a infant formula was to evaluate the possible influence and ensure the dissolved in 10 mL water of LC-MS grade and accuracy of analysis. aliquots were stored at -80 °C freezer in amber tubes Method Validation until analysis. Pooled breast milk which mixed 2 L human milk samples was also used as the quality The method was validated by linearity, limit of control material for each analytical process. 5 detection (LOD), limit of quantification (LOQ), replicates of the NIST samples and the pooled breast accuracy, precision, recovery, matrix effects and milk were analyzed with each run. stability in accordance with ‘Guideline on bioanalytical method validation’ of the European Sample Pretreatment Medicines Agency[36] and Commission Decision of [35] Breast milk samples stored at -80 °C and were the European Communities .