Vitamin D3 and related compounds by ESI and APCI

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1 Liquid Chromatography Mass Spectrometry SSI-LCMS-9 Vitamin D and related compounds by ESI and APCI LCMS-8 Summary Vitamin D and related compounds were measured by LC-ESI/APCI-MS-MS. Background Accurate and sensitive measurement of vitamin D and related compounds require approaches such as UHPLC-MS-MS. Isotopically labeled internal standards are an important part of such trace quantitative methods. In this analysis, Vitamin D, its metabolite -OH-D, and corresponding deuterated internal standards were measured using a Nexera UHPLC with an LCMS-8 triple quadrupole MS detector with both ESI and APCI ionization. Method Authentic standards in solution were diluted in methanol to optimize instrument parameters and construct calibration curves. A Phenomenex Kinetex C8 column (.6 µm,. mm) was used with a binary gradient of mm ammonium bicarbonate (Pump A) and methanol (Pump B). The flow rate was. ml/min and the column temperature was C. The injection volume was µl. ESI was used for ionization in positive mode. The nebulizing gas was L/min, the interface temperature was C, the heater block and DL temperatures were C, the drying gas was L/min, and the heating gas was L/min. Analyte separation was accomplished isocratically at 97% B. vitamin D hydroxyvitamin D Figure : Structures of Vitamin D and hydroxyvitamin D.

2 SSI-LCMS-9 APCI was used for ionization in positive mode. The nebulizing gas was L/min, the interface temperature was C, the heater block and DL temperature were C, and the drying gas was L/min. Protonated molecules and their respective singly dehydrated ions were selected as precursor ions, and a wide spectrum of product ions were considered for MRM analysis. For each compound, transitions were chosen based on overall performance characteristics including signal intensity and background level. Results and Discussion Calibration curves over the range of.6 to ng/ml (ESI) and.9 to ng/ml (APCI), all run in triplicate, were linear as shown in Figures and. Representative chromatograms are shown in Figures and. Figures and 6 demonstrate a complete acquisition with.6 pg and a blank injection. Table provides limits of quantitation for each of the analytes dependent on ionization type. The relative standard deviation of LLOQ standards were less than % (APCI) and % (ESI) with signal to noise greater than ten. Area (x,,) Vitamin D r = Area (x,,) -OH Vitamin D r = Conc. Area (x,,) Vitamin D- H r =.9999 Conc. Area (x,,) -OH Vitamin D- H 6 r = Conc. Conc. Figure : Calibration curves for each compound using ESI ionization.

3 SSI-LCMS (x,) :8.>9.(+) :8.>7.(+) Vitamin D.6 ng/ml.7 pg in. µl (x,) :.>8.(+) :.>6.(+) :.>.(+) -OH Vitamin D.6 ng/ml.7 pg in. µl (x,) :88.>7.6(+) Vitamin D- H :88.>9.(+) :88.>6.(+).6 ng/ml :88.>8.(+) :88.>6.(+).7 pg in. µl :88.>6.(+) (x,) :7.>89.(+) :7.>7.(+) :7.>.(+) -OH Vitamin D- H 6.6 ng/ml.7 pg in. µl Figure : Representative chromatograms of each standard from ESI ionization. Area(x,) Vitamin D 7. r =.998 Area(x,) -OH Vitamin D. r = Conc. 7.ISODx.lcb. Conc. 7.ISODx.lcb Area(x,) Vitamin D- H r = Area(x,) -OH Vitamin D- H 6 r = Conc. Conc. 7.ISODx.lcb Figure : Calibration curves for each compound using APCI ionization. 7.ISODx.lcb

4 SSI-LCMS-9 (x,) :8.>67.(+). :8.>9.(+) :8.>.(+) :8.>7.(+).. Vitamin D 7.8 ng/ml. (x,) :.>8.(+) :.>6.(+). :.>9.(+) :.>9.(+) :.>7.(+). :.>.(+).7 -OH Vitamin D 7.8 ng/ml ISODx_9_DL9_9.lcd ISODx L9_.lcd (x) :88.>7.(+) :88.>8.(+) :88.>6.(+) :88.>6.(+) 7. :88.>6.(+) :88.>9.(+) Vitamin D- H.9 ng/ml (x) 6. :7.>89.(+) :7.>7.(+). :7.>9.(+) :7.>.(+) :7.>.(+). :7.>9.(+) -OH Vitamin D- H 6.9 ng/ml ISODx_8_DISL_8.lcd 7.ISODx ISL_.lcd Figure : Representative chromatograms of each standard from APCI ionization. (x,) :-OH Vit. D.>8.(+) CE: -. :-OH Vit. D.>.(+) CE: -. :-OH Vit. D.>6.(+) CE: -. :-OH Vit. D[H6] 7.>89.(+) CE: -. :-OH Vit. D[H6] 7.>7.(+) CE: -. :-OH Vit. D[H6] 7.>.(+) CE: -. :Vit D 8.>9.(+) CE: -6. :Vit D 8.>7.(+) CE: -8. :Vit. D[H] 88.>7.6(+) CE: -. :Vit. D[H] 88.>9.(+) CE: -. :Vit. D[H] 88.>6.(+) CE: -7. :Vit. D[H] 88.>8.(+) CE: -7. :Vit. D[H] 88.>6.(+) CE: -. :Vit. D[H] 88.>6.(+) CE: min Figure : Representative chromatogram of each standard from ESI ionization with.6 pg in µl injection.. :-OH Vit. D.>8.(+) CE: -. :-OH Vit. D.>.(+) CE: -. :-OH Vit. D.>6.(+) CE: -. :-OH Vit. D[H6] 7.>89.(+) CE: -. :-OH Vit. D[H6] 7.>7.(+) CE: -. :-OH Vit. D[H6] 7.>.(+) CE: -. :Vit D 8.>9.(+) CE: -6. :Vit D 8.>7.(+) CE: -8. :Vit. D[H] 88.>7.6(+) CE: -. :Vit. D[H] 88.>9.(+) CE: -. :Vit. D[H] 88.>6.(+) CE: -7. :Vit. D[H] 88.>8.(+) CE: -7. :Vit. D[H] 88.>6.(+) CE: -. :Vit. D[H] 88.>6.(+) CE: min Figure 6: Representative chromatogram of a blank from ESI ionization with µl injection.

5 SSI-LCMS-9 Limit of Detection Vitamin D Vitamin D -[ H ] -OH Vitamin D -OH Vitamin D -[ H 6 ] Ionization ESI APCI <.7 pg 7.8 pg <.7 pg.9 pg <.7 pg 7.8 pg <.7 pg.9 pg Table : Limits of Quantitation for ESI and APCI ionization. Conclusion Vitamin D and related compounds were analyzed using the LCMS-8 and APCI for high sensitivity quantitation.

6 LCMS-8 LCMS-8 LCMS-8 LCMS- LCMS-IT-TOF Founded in 87, Shimadzu Corporation, a leader in the development of advanced technologies, has a distinguished history of innovation built on the foundation of contributing to society through science and technology. Established in 97, Shimadzu Scientific Instruments (SSI), the American subsidiary of Shimadzu Corporation, provides a comprehensive range of analytical solutions to laboratories throughout North, Central, and parts of South America. SSI maintains a network of nine regional offices strategically located across the United States, with experienced technical specialists, service and sales engineers situated throughout the country, as well as applications laboratories on both coasts. For information about Shimadzu Scientific Instruments and to contact your local office, please visit our Web site at Shimadzu Corporation SHIMADZU SCIENTIFIC INSTRUMENTS, INC. Applications Laboratory 7 Riverwood Drive, Columbia, MD Phone: Fax: -8- URL For Research Use Only. Not for use in diagnostic procedures. The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu. The information contained herein is provided to you as is without warranty of any kind including without limitation warranties as to is accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication. This publications is based upon the information available to Shimadzu on or before the date of publication, and subject to change without notice. Shimadzu Scientific Instruments, First Edition: October,

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