Cee ean eects Dieses ict aI MLN INTRODUCTION Vitamins are ntvents essential to human health, andar present in almostall types ffods.in ation o food sources, vitamin supplements fon consumed to ensue adequate aint cu ns owe sullen vitamin tobias andor mito inthe choice of fooes, or maltunctons in digestion and ingestion. Vitamin supplements are avaliable in various forms such as single- or multivitamin tablets, formula, and vitmin- eich eve ot comercial ft tied wt vitamins andor oternuttionl essentials such as minerals. Based on ther solubility, vitamins are divided int vo categories: water-soluble vitamins (WSV) and fat-soluble vitamins (FSV). WSVS include vitamin C (ascorbic acid, B, (thiamine), B, (tte), , (niacin, niacinamide), B, (pentathenic acid), 8, (pyrodoxine),B biotin, 8, ois acid), and, (Gyanocobalamine). Accurate quantitative measurements for vitamins are required to ensure product quality and regulatory compliance as well as to monitor vitamin nak. r Established methods for vitamin analysis include microbiological ‘mathads, which are typically designed for single vitamin analysis and are time consuming, “and chromatographic methods, including gas, chromatography capillary electrophoresis, and liquid chromatography (LC) with various methods of detection." a vty of matrices wth various mades of deletion "*""-" Here we present a high throughput mend for simultaneous determination for the above mentioned ten WSVs using utrahigh-pertormance LC and tandem mass spectrometry (UHPLC-MS/MS). Chromatography was optimized forthe total resolution ofall target analytes ona Thermo Scie Dio Aslam C30 reversed-phase (RP) column. An MS) IMS instrument was operated in selected reaction monitoring (SRM) ‘mode forthe best selctiy and sensitivity, and an isotope labeled internal standard (Std) was used for accurate quantitation Randomly selected VEBs were assayed by this method forthe selected vitamins, and when compared with product labeling, much higher values, were observed for mast ofthe vitamins. EXPERIMENTAL Chemicals and Reagents ‘A set of WSY standards was purchased from AccuStandard (P/N \IT-WSK-R1-SET) conlaining ten individual chemicals. sotope labeled intemal standard pydoxine-d, was purchased trom C/DYN Isotopes (PIN: D-6819). Ammonium formate and fomic acd were purchased ‘om Sigma-Aldrich, Acetonitrile was oblained trom Burdick & Jackson (HPLC/UV grade). Deionized water (DIH,0) was wilt 0 @ Milipore water station Preparation of Standards Individual stock solutions were prepared by aissoving an appropiate amount of purechemial in 1% formic aida! my (1000pas- per milion, po s rnd. F by anmonta, 4% and0 respective) The was prepared n 1% formic acid at 10 ppm to prepare calibration standards and spike unknown samples. Calibration standards were prepared in 0.1% formic acid and ranged ‘rom 10 parts-per-bllion (ppb) to 5000 ppb at 7 levels: 10 ppb, 50 ppb, 100 ppb, 500 ppb, 1000 ppb, 2000 ppb, and 5000 ppb Target analytes ‘were divided into three groups: Group 1 containing only B,; Group 2 containing B, and B,,: and Group 3 containing B,, 8, (niacin and niacinamide), Band, IStd was spiked in each calibration standard at 500 pab. Preparation of Vitamin-Enriched Beverage Samples ‘EB samples were randomly selected and purchased from a local {grocery store and kept at room temperature until analysis. Carbonated \VEBs were degassed using a sonication bath for 30 seconds. A mL aliquot of each sample was transteredtoa1.5 mL autosampler val, spiked with Std at 500 ppb vortex med, and analyzed for Group 1 and Group 2 vitamins. A10 ul alquot ofeach sample was pipetted to another 1.5 mL autosampler via, diluted with 990 pL DI HO, spiked with IStdat 500 ppb vortex mixed, and then analyzed for Group 3 vitamins.UHPLC-MS/MS Analysis, med using @ Therma Sent Table 2. SRM MS/MS Events and Parameters ae fe Retention [cot gystem coupled wth a Thermo Scns " TSO Quantum Access MAX” MS/MS instrument via a heated ots ah ana electrospray ionization (HES!) source. Chromatographic separation was achieved using a Acclaim C30 column (P/N: 075725, 2.1 x 150 mm, 3 ‘Ascorbic Acid cl] 12 a pi), Mobil phase consisted of three components: A) 10 mM formate 15 | 8 buffer (pH 4.0; B) 10mM formate bute (pH 3.0); and C) 90% CH,CN, w | 2 10% 10 mM formate butter (pH 3.0) Gradient elution was used with a B17 |S 1h lino deta liste in Table . Flow rate was seta 0.6 mL/min an the column ‘temperature was set at 15 °C. The MS/MS instrument was operated in Thiamine a) 30 |2s47| 25 21 SRM mode wit he details listed in Table 2. The HES| ionization source : 18 parameters were set as follows: Spray Voltage (4000 V); Vaporizer im pa Temperature (250°C); Capillary Temperature (200 °C); Sheath Gas (40 Pionne so Jaro] 1m arbitrary units) and Auliary Gas 60 arbitrary units) 5 1Si:Pyodoried, {st 50 |4r-ro] 172 4 wm | 8 wo | 2 Pee Niacin 58 [47-70] 3 P= Time a 8 c wf 7 00 7 7 Panohesicted |a,] 72 Jro-g0] 20 | a0 100 0 a oe [iw [ar 5 a0 a 7 Cyanccotaine 8] 106 foo-tsop—— + 35 a 100 a a | a0 7 7 a Folic Ai 8] 109 fool wo POO at a 20 ® war | ig 7 a a Biotin a] m2 poral 26 -EE 150 700 0 a 2 | 8 Fist 8] 7 fossa) av 23 [21 RESULTS AND DISCUSSION Chromatography ‘Although many LC methods have been reported for simultaneous analysis ‘of WSs, these methods usually suf tom low traughput or incomplete chromatographic resolution, and several highly hydrophilic analytes are poorly retained onthe commonly used C18 RP columns. In this study, a 30 column was used to improve the retention of poorly cetaned analytes, suchas vilamin C and thiamine. In ation, ammonium formate was buffered at wo pl condtions: pl 3.0 and pH 4.0, withthe higher pH butter used in the early phase ofthe gradient to further improve thertention for thiamine, andthe lower pH bufer used to provide complete resolution for leer eluted vitamins, Under the optimized conditions al target vitamins were baseline separated within 12 min. The minimum retention factor was observed for vitamin Cat 1.3 (retention time 1.2 min), and the retention factor t ain was obse'ved at £5, which was gr ficantty imotoved aver ously ebotedmetoes wre than elec frst wi ee tir fcr fst 1" Varn and, aa WV hoop and were nt visible inthe UV chromatogram. However, the peak labels for both analytes ae show in Figure 1A to demonstrate the chrometographic separation. These two analytes were detected by MS} [MS with great senstvy s seen in Figure 28 and Figure 2C 2 Simultaneous Analysis of Water-Soluble Vitamins in Beverages by UHPLC-MS/MS‘rman Catan erspectaner non Seer mee awesc | See etree Sree seein o Sera. oe Tee BE sae | - 6.00 0382 aes [Eo Ea [| fe | fe = mi ao Figure 14) chromatograms of al target WSUS. 8) O-SAIM chromatograms of Group § and 2 vias. C)0-SAM cromaograns of Group 3 vans Mass Spectrometry Electrospray ionization (ES!) was used inthis study asthe ionization interface de ots suitability and beter sensiity for polar compounds than other atmospheric pressure ionization (AP) techniques. lnization souroe parameters forthe HESI probe used in his study were optimized to provide best sensitty and were described in the Experimental section, Under the optimized chromatographic and ionization conditions, most analytes exhibited strong protonated molecular ions (i, [MsH) excep for vitamin C and folic acid where deprotorated molecular ions [M-H]- were observed as the dominant MS peak. A trong doubly-charged MS peak was observed fr vitamin B,, a 679 ‘mz wells the [MH] at 1356 m/z For each analy, the two most intense fragments were selected asthe monitored produc ions, which are listed in Table 2 along with the optimize colsion energies. Thus foreach analy, two SRM transitions were monitored with one being ‘quantitative SAM (Q-SRM), which showed relatively stronger MS response, arth olar oe ng contmaive SAM (C-SAM) The 1-SRM chromatograms are shown in Figure 18 and Figure 1C, with each of the vitamins at 50 ppb. The MS/MS detection demonstrated great sensitivity and selectivity fr vitamin analysis even at low pp levels 'AL50 ppb, the minimum signal-to-noise ratio (SiN) was observed at 20 for niacin (26 for niacinamide) withthe rest ofthe target analytes showing SIN greater than 100. The great senitty provided by MS/MS instrumentation enables the quantitation for low level vitamins suc a8 8, and flic acid in complex matrices, which were not achievable with previously reported methods using only UV detection Quantitation (One ofthe challenges encountered in his study was the large diterences in concentration o the vitamins present in beverages or tablets. the tested samples, the concentrations of Group 3 vitamins (mg levels per serving) were roughly 1000 times the concentrations of Group 1 and 2 vitamins (ug levels per serving). The lowest concentration observed was 0.6 wg per serving (B,) while the highest concentration was at 20 mg pe serving (8). Asingle assay trying to cover the whole concentration range is beyond the linear response range of any mass spectrometer. Two approaches are usualy practiced to address this challenge among reprted methods covering these wide concentration anges. Some reported methods use MS fr lower concentration analytes and less sensitive detectors such 25 UV forthe quantitation of high concentration vitamins, thus lasing the selectivity of MS quantitation and may utter rom interferences and/or lower quantitation accuracy. Another approach performs several assays for each sample with diferent dilution factors and results are reported wit the most appropriate ciltion. In this study, te ater approach was used and two assays were performed: the primary one quantitating low concentration vitamins including BB, and, which were assayed directly ater spiking intemal standard; and the secondary assay quantitating the remaining vitamins a higher concentrations ater 4 10-fold ition and espiking the cuted sample with ISt o 500 foo This technique too ul advarcage of Ie selectivity ane soeciity Provided by MIS detection thus ensuring quantitation accuracy.Stability of vitamins in solution was another challenge. Vitamin C was estremely unstable in muiiitamin solutions, and degradation was observed within 20 min eventhough the sample was prepared in acdc solution and placed ina thermostatted autosampler at 4°C. istbility of he analyte tse can cause substantia variance inthe quantitative determination of vitamin C, and thus it was not included for quantitation inthis study Instability was also reported for other vitamins, such as ribotiain, pyridoxine, a thiamin, which ae ligt sensi," and thiamin, pentothenic acid (in acid or basic condition) folic ci, and Pyridoxine, which are heat labile. To avoid oss of analytes during analysis, samples wee prepared in amber autosampler vials and promptly placed in the rtrigeated autosampler a 4 °C. An addtional challenge for accurate quantitation was the interactions of vitamins when present together in solution. Interactions between B, tolicacid, and botevin have teen pare" thus targeted lamin were dived int three groups wih additional consideration of thelr concentrations in samples: Group 3 included higher concentration vitamins (8, B, 8, and B,) and Group 2 included lower concentration vitamins (8, and). hough folic acid was also present in lover concenration in samples and could be included in Group 2 vitamins, observations revealed that quantitation of lw concentration folic acd oul ly ela w by theo B, and... Thus thre calibration standard ses were prepared forthe tree groups of vitamins to generate individual calibration curves for quantitation Method Performance Method performance was ev cf determination, precision, and acura, Caliban cures foreach analyte were generated fom calibration standards with concentrations fo tos Is 2 used fT the experimental ata ard was sed 25th weighting factor. Detaled resul 3. Evale cooticirt lat precision, nd accuray were achieved for each target vitamin. Limits of quantitation (LOs) were determined a the lowest concentration in calibration standards exiting signal-to-noise ratios (S/N) greater than 10. LOQ was observed at 10 ppb for most analytes, excep for niacin and niacinamie at 50 pob, and folic acid at 100 ppb. Although SiN for folic acid was achieved with values much greater than 10 at lower concentrations, por quantitation accuracy was observed, which was believed to be the reduced stbity ofthis analyte when present isis ore Th etd i sy f cuartfication of toe it s iis thd, proven byte Sales observed at LOO. Howee this ‘method was designed and the calibration range set to minimize sample preparation procedures, number of dilutions, and assays tobe run in oder to maintain a high analytical throughput. Fable 3. Calibration, Coefficient of Determination, Precision, Accuracy and Detection it 50 ppb 2000 po Anaine ce 8, [Nasi soso [came [as [ou | a2 | 0 | 3% | 0 | wom 8, | Thanine “o-soo0 [ove [20 [30 | eo | emo | 2 | ims | wea 8, | rile 70-5000 [0906 [a [3m | aoe | 1s [ae | 32 | voerom) 8, [_ Neate so-soo0 [0909 [0 | 3m | 19 |e | 20 | a2 | wor 8, | Paiatencact | s0-2m0 | osooa [ar | as7_| eae | 102 | sar | ot | 106100) 8, | Pyridoxine osm [100 [soa [iz | 1 | 190 [18 | ro | 10680) 8, | Bian 70-5000 | oa%es [re | az | ee [190 [as | 55 | tocr000) 8, | Folic aaa voo-soo0 | oaea [a [ase [113 | 19% [a3 | ora | soocotomoy Cyanocotsranine | 10-000 | _oserr [a7 | 102 [ss | 1s [aot | esa | 10(>1000 “Al ecion ad aca sus were sunmarizdom sven repeat assays “Prison ard azz eu fr lal otal am 100 pan 2000p tarda. 4 Simultaneous Analysis of Water-Soluble Vitamins in Beverages by UHPLC-MS/MSAnalysis of Vitamin-Enriched Beverages Samples ‘As described inthe Experimental section, ten beverage samples were selected and analyzed for tei vitamin content. Among te selected bev viami-ored wat Ive were vtamin-ere creo ene clink, The results are shown in Table 4. Large diferences were ‘observed between measured and labeled values, and this observation agreed with previously conducted studies ®* An explanation for these descrepancis coule te fal te k rormad tk higher than label claims, deviating inthe direction of no harm: to ‘compensate for extrapolated degradations during slorage and ste lite ea Gene eat This study describes a UNPLC-MS/MS method for simultaneous uaniation of WSV in beverages. Tis method demonstrated excellent correlation of determination, precision, accuracy and selective and sensitive detection with low quantitation its. This matod was successfully applied to the determination of WSVS in beverages and energy supplements with presented results. einer ‘Analyte vest | vee2 | vees | vena | vees | vee | vee7 | veee | veeo | veow0 Fito 22007) 53a [ 1407) Nicatinarie s7@ | 2a | 30 [26a | 2@ | eo [ wen | en [2600 [ aon Parohenicacd [as [ toy [ 3@ | 35m | asa | 5306) zai) | 24g) [ 3925) Pyrdorne sijoay [03502 [0704 | oso | ison [ 63s) | 2a@ | sea | aia | sses) Cyanocobalanine 47a [36a [ sa | 79@ [som [ites Label vals of vitins are ncudedinparenteses Duplicate assays were performed foreach sampleREFERENCES % 1. Davis, RE; Moulton, J; Kelly, A An Automated Microbiological Method forthe Measurement of Vitamin ,,. Journal of Cinial Pathology 1978, 26(7), 494-496. 2. Boke, H.; Sobota, H, Microbiological Assay Methods for Vitamins. in Advances in Ciinical Chemistry Hay, S.; Stewart, C. P, Eds. Elsevier 1963; Vol. 5, pp 173-236, 3. Kory, W. Gas Chromatography of Vitamin B, In Methods in Enzymology, Donald, B. 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