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LC/MS/MS Quantitative Analysis of Water Soluble in Blood

Lauren Frick, Rory Doyle MSACL 2015 Agilent Technologies, Inc, Lexington, MA and Wilmington, DE Poster No.- 21

Introduction Experimental Results and Discussion The major water soluble vitamins include B1 (, Thiamine Sample Preparation Pyrophosphate), Vitamin B2 (), Vitamin B3 (Nicotinic Acid and ), • 200 µl of serum sample, calibrators, controls was taken and 20 µl ISTD at 1000 Table 2- Sensitivity Vitamin B5 (Panothenic Acid, Pantothenol and Pantethine), (, ng/ml were added to each µ Compound LOD LOQ Compound LOD LOQ Pyridoxal, and Pyridoxal-5-Phosphate), Vitamin B7 (), Vitamin B9 (Folic • 100 l of 5% Trichloroacetic Acid was added to each tube and vortexed for 5 (ng/mL) (ng/mL) Acid, and 5-Methyltetrahydrofolate) and Vitamin B 12 (, minutes prior to centrifugation for 10 minutes at 13000 rpm • 25 µl of 1N Sodium Hydroxide was added to quench the acid and prevent damage , and ). Thiamine 0.1 0.25 Pyridoxine 0.1 0.25 to the column Therefore, we developed LC/MS/MS analytical methods and evaluated various • The supernatant was transferred to MS vials for analysis Thiamine pyrophosphate 10 25 Pyridoxal-5-Phosphate 10 25 columns and solvent combinations in order to demonstrate the chromatographic All in-house calibrators were prepared in DC Mass Spec Gold Serum (Golden West separation, detection and quantification of the water soluble vitamins in blood. The Biological, Inc.) Riboflavin 5 10 Biotin 0.25 0.5 sample preparation was a simple protein precipitation and the methodologies were Method developed on an Agilent 1260 HPLC and 6460 Mass Spectrometer with a 8 minute Nicotinic Acid 0.1 0.25 Folic Acid 5 10 analytical gradient method in positive ionization mode. HPLC Conditions Agilent 1260 Infinity HPLC series binary pump, well plate, thermostatted column compartment Nicotinamide 0.1 0.25 Folinic Acid 25 50 Column: Agilent Technologies Poroshell 120, SB-AQ, (or Extend) 2.5 5 10 25 B1 3 x 100 m, 2.7 µm 5-Methyltetrrahydrofolate Agilent Technologies Poroshell 120 Fast Guard 2.1 x 5 mm Pantothenol 0.25 0.5 Cyanocobalamin 2.5 5 Column Temperature: 25 oC Injection Volume: 5 µl Pantethine 1 2.5 Adenosylcobalamin 5 10 Autosampler Temperature: 4 oC Needle Wash: Flush port (50%Methanol:50%Water) 5 seconds Pyridoxal 0.25 0.5 Methylcobalamin 5 10 B2 Mobile Phase A: 0.1% Formic Acid+5mM Ammonium Formate Water Mobile Phase B: 0.1% Formic Acid in Methanol Pyridoxamine 0.1 0.5 Hydroxocobalamin 0.1 0.25 Flow Rate: 0.5 ml/min Gradient: 0 min- 100%A:5%B Table 3-Chromatograms 6 min- 5%A:95%B Run/Stop time: 8 minutes/3 minutes

MS Conditions B3 Agilent 6460 Triple Quadruple Mass Spectrometer- Dynamic MRM Ion mode: Agilent Jet Stream Positive Mode B9 Gas Temperature: 300oC Gas Flow: 8 L/min Nebulizer: 38 psi B5 Sheath Gas Temperature: 400o0C Sheath Gas Flow: 112 l/min Capillary Voltage: 2100V Nozzle Voltage: 0V Q1/Q2 Resolution: 0.7/0.7 unit Cpd 1: Adenosylcobalamin: +ESI MRM Frag=152.0V CF=0.000 DF=0.000 [email protected] (790.3 -> 147.1) TCA 100-100.d x10 5 Delta EMV/CAV: +400V/2 1 1 4.936 415196 0 Cpd 2: Cyanocobalamin: +ESI MRM Frag=152.0V CF=0.000 DF=0.000 [email protected] (678.2 -> 147.1) TCA 100-100.d x10 5 Table 1: MRM Acquisition Table- * Quantifier Ion 1 1 4.417 3230136 0 Cpd 3: Methylcobalamin: +ESI MRM Frag=132.0V CF=0.000 DF=0.000 [email protected] (673.0 -> 147.1) TCA 100-100.d x10 5

Compounds R MRM Fragmentation Collision 1 1 5.068 512884 4.094 32533 (Min) Voltage Energy 0 Cpd 4: Hydroxocobalamin: +ESI MRM Frag=157.0V CF=0.000 DF=0.000 [email protected] (665.0 -> 147.1) TCA 100-100.d x10 6

1 1 Thiamine 1.44 265.1 – 144/122* 97 8/12 3.939 18585247 0 Cpd 7: Pantothine: +ESI MRM Frag=147.0V CF=0.000 DF=0.000 [email protected] (555.2 -> 425.1) TCA 100-100.d x10 6 Thiamine Pyrophosphate 1.19 425.1 – 122.1* 122 44 1 1 4.725 20139126 0 Cpd 8: Folinic Acid: +ESI MRM Frag=122.0V CF=0.000 DF=0.000 [email protected] (474.2 -> 327.1) TCA 100-100.d Thiamine-13C4 1.42 269.2 – 122.1 82 12 x10 4 1 1

3.452 83067 4.434 100838 6.218 37615 B6 Riboflavin 4.48 277.2 – 243.1*/198.1 167 20/40 0 Cpd 9: 5-Methyl-Tetra-Hydrofolate: +ESI MRM Frag=122.0V CF=0.000 DF=0.000 [email protected] (460.2 -> 313.1) TCA 100-100.d x10 4

1 1 Riboflavin-13C4,15N2 4.45 383.2 – 202.1 167 40 3.721 196909 5.370 22208 0 Cpd 11: Folic Acid: +ESI MRM Frag=102.0V CF=0.000 DF=0.000 [email protected] (442.2 -> 295.1) TCA 100-100.d x10 5

Nicotinic Acid 1.72 124 – 80.1*/53.1 107 20/32 1 1

4.543 922708 0 Cpd 12: Thiamine Pyrophosphate: +ESI MRM Frag=122.0V CF=0.000 DF=0.000 [email protected] (425.1 -> 122.1) TCA 100-100.d Nicotinic Acid-13C3,15N 1.69 128.1 – 54.1 102 36 x10 4

1 1

1.190 113648 1.666 6269 2.016 9277 2.311 8172 Nicotinamide 2.31 123.1 – 80.*1/53.1 112 20/32 0 Cpd 14: Riboflavin: +ESI MRM Frag=167.0V CF=0.000 DF=0.000 [email protected] (377.2 -> 243.1) TCA 100-100.d B7 B12 x10 5 Nicotinamide- 13C3,15N 2.29 127.1 – 83.1 112 20 1 1 4.484 1150311 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 2.2 2.4 2.6 2.8 3 3.2 3.4 3.6 3.8 4 4.2 4.4 4.6 4.8 5 5.2 5.4 5.6 5.8 6 6.2 6.4 6.6 6.8 7 7.2 7.4 7.6 7.8 Pantothenic Acid 2.39 220.1 – 90.1*/72.1 107 12/20 Counts vs. Acquisition Time (min)

Cpd 16: Thiamine: +ESI MRM Frag=97.0V CF=0.000 DF=0.000 [email protected] (265.1 -> 122.1) TCA 100-100.d x10 6 Pantothenic Acid-13C3,15N 2.36 224.1 – 94.1 97 12 1 1 1.441 105271765 1.941 7876346 0 Cpd 18: Pyridoxal-5-Phosphate: +ESI MRM Frag=152.0V CF=0.000 DF=0.000 [email protected] (247.9 -> 150.0) TCA 100-100.d x10 4

Panthothenol 2.36 206.1 – 76.1/58.1 92 8/20 1 1

1.497 126158 0 Cpd 19: Biotin: +ESI MRM Frag=102.0V CF=0.000 DF=0.000 [email protected] (245.1 -> 227.1) TCA 100-100.d Pantethine 4.73 555.1 – 425.1/295.1 147 8/20 x10 6

1 1

4.130 12856301 Experimental Pyridoxamine 1.29 169.1 – 152.1*/134.1 82 8/20 0 Cpd 21: Pantothenic Acid: +ESI MRM Frag=107.0V CF=0.000 DF=0.000 [email protected] (220.1 -> 90.1) TCA 100-100.d x10 5

1 1 Reagents, Standards, Calibrators and Controls Pyridoxamine-2H3 1.26 172.1 – 155.1 82 8 2.390 2775419 0 Cpd 22: Pantothenol: +ESI MRM Frag=92.0V CF=0.000 DF=0.000 [email protected] (206.1 -> 76.1) TCA 100-100.d Standards/Calibrators x10 6 Pyridoxal 1.58 168.1 – 150.1*/94.1 82 24/28 1 1 2 Biotin: Isosciences Biotin- H : Isosciences 2.364 33465255 8 0 Cpd 26: Pyridoxine: +ESI MRM Frag=92.0V CF=0.000 DF=0.000 [email protected] (170.1 -> 152.1) TCA 100-100.d 2 Pyridoxal-2H3 1.55 171.1 – 153.1 87 8 x10 7 Pyridoxal: Isosciences Pyridoxal- H3: Isosciences 1 1 2 1.654 62345867 Pyridoxine: Isosciences Pyridoxine- H : Isosciences Pyridoxine 1.65 170.1 – 152.1*/134.1 92 8/20 0 3 6 Cpd 27: Pyridoxamine: +ESI MRM Frag=82.0V CF=0.000 DF=0.000 [email protected] (169.1 -> 152.1) TCA 100-100.d 2 x10 Pyridoxamine: Isosciences Pyridoxamine- H : Isosciences 1 1 3 Pyridoxine-2H3 1.62 173.1 – 155.1 97 12 1.299 357948691.649 16296387 13 15 0 Cpd 28: Pyridoxal: +ESI MRM Frag=82.0V CF=0.000 DF=0.000 [email protected] (168.1 -> 150.1) TCA 100-100.d Riboflavin: Isosciences Riboflavin- C4, N2: Isosciences x10 6 13 Pyridoxal-5-Phosphate 1.49 247.9 – 150/94.1 142 12/28 1 1 1.581 34404103 Thiamine: Isosciences Thiamine- C : Isosciences 3.433 6129103 4 0 Cpd 31: Nicotinic Acid: +ESI MRM Frag=107.0V CF=0.000 DF=0.000 [email protected] (124.0 -> 80.1) TCA 100-100.d 13 15 Biotin 4.13 245.1 – 227.*1/123 102 12/28 x10 5 Pantothenic Acid: Isosciences Pantothenic Acid- C3 N1: Isosciences 1 1 13 15 1.716 2686334 2.312 571301 Nicotinamide: Cerilliant Nicotinamide- C , N : Isosciences Biotin-2H8 4.11 253.2 – 235.1 122 8 0 3 1 Cpd 32: Nicotinamide: +ESI MRM Frag=112.0V CF=0.000 DF=0.000 [email protected] (123.1 -> 80.1) TCA 100-100.d 13 15 x10 6 Nicotinic Acid: Cerilliant Nicotinic Acid- C3, N1: Isosciences 1 1 Folic Acid 4.54 442.2 – 295.1*/176 102 12/40 2.311 36849003 13 15 0 Folic Acid: Sigma Folic Acid- C5, N1: Isosciences 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 2.2 2.4 2.6 2.8 3 3.2 3.4 3.6 3.8 4 4.2 4.4 4.6 4.8 5 5.2 5.4 5.6 5.8 6 6.2 6.4 6.6 6.8 7 7.2 7.4 7.6 7.8 Folinic Acid: Sigma-Aldrich Folic Acid-13C5,15N 4.52 448.2 – 295.1 117 12 Counts vs. Acquisition Time (min) 5-Methyltetrahydrofolate: Cayman Chemicals Folinic Acid 4.43 474.2 – 327.1/120 122 16/40 Cyanocobalamin Cerilliant 5-Methyltetrahydrofolate 3.72 460.2 – 313.1/180 122 16/48 Adenosylcobalamin : Sigma-Aldrich Conclusions Methylcobalamin: Sigma-Aldrich Cyanocobalamin 4.42 678.2 – 359.1/147.1 152 20/40 Hydroxocobalamin: Sigma Aldrich Adenosylcobalamin 4.94 790.3 – 147.1/227.1 152 32/88 • Baseline separation of the water soluble vitamins was achieved within a 8 minute Pantothenol: Sigma-Aldrich Methylcobalamin 5.06 673 – 359.1/147.1 132 20/48 Pantethine: Sigma-Aldrich run on a Poroshell 120 SB-AQ column. Hydroxocobalamin 3.99 665.1 – 359.1/147.1 157 20/40 • Excellent linearity (>998) of calibration curves with great accuracy, precision and Thiamine Pyrophosphate: Sigma-Aldrich reproducibility was achieved for all vitamins Pyridoxal-5-Phospahte: Sigma-Aldrich • Further evaluate different sample preparation techniques and water soluble vitamin Linearity free serum matrixes available to determine which gives the best results while Reagents The assay was linear over the calibration curve from 0.1 to 5000 ng/mL with a maintaining low cost and ease of use. Methanol: Honeywell Ammonium Formate: Sigma-Aldrich mean of coefficient of determinations (R2) > 0.998 and %CV <10%. Standards • Further evaluate the different Water Soluble Vitamins per a group method rather Formic Acid: Sigma-Aldrich Trichloroacetic Acid: Sigma-Aldrich need to be made fresh in water and away from light at all times to avoid than a global method for better analytical needs, chromatography and results. Sodium Hydroxide: Sigma-Aldrich oxidation. For research use only. Not to be used for diagnostic procedures