Integration of amino acid, acylcarnitine and steroids analysis in single FIA/LC-MS/MS platform

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1 POCON3E Integration of amino acid, acylcarnitine and steroids analysis in single FIA/LCMS/MS platform ASMS 2 ThP2 Tetsuo Tanigawa, Toshikazu Minohata Shimadzu Corporation, Kyoto, Japan

2 analysis in single FIA/LCMS/MS platform Introduction Analysis of amino acids (AA) and acylcarnitines (AC) in dried blood spot (DBS) sample collection method by flow injection analysis (FIA) is now widely used. On the other hand, traditionally, analysis of steroid such as 7hydroxyprogesterone is done by immunoassays but LC/MS/MS will be an attractive analytical alternative because it can also screen for other related steroids. The use of LC/MS/MS results in a reduction of false positives and a more accurate quantitative performance. The requirements against steroid analysis by LC/MS/MS are getting more stringent issues. In this study, we present a strategy for performing both AA/AC and steroids analysis within a single LC/MS/MS platform. Methods and Materials The experimental setup designed to combine a FIA measurement covers 8 AAs and 7 ACs and a LCMS measurement for steroids includes cortisol, 2deoxycortisol (2DOF), deoxycortisol (DOF), androstenedione (4AD) and 7hydroxyprogesterone (7OHP) with 2 position 6 port high pressure valve. AAs, ACs Guard LC3AD SIL3AC F2AH LCMS (LCMS) F2AH Steroids Guard Guard (SIL3AC) SIL3AC LC3AD F2AH LCMS Figure Flow Diagram of FIA/LCMS/MS system 2

3 analysis in single FIA/LCMS/MS platform The isotopically labeled internal standards for amino acids, acylcarnitines, and steroids were purchased from Cambridge Isotope Laboratories, Inc. Quality control materials were obtained from the Newborn Screening Quality Assurance Program at the Centers for Disease Control and Prevention (CDC). Analytical Conditions HPLC Mobile Phase A :. Formic acid water Mobile Phase B : Methanol temperature : 4 ºC [for Amino Acids and Acylcarnitines] Guard : GL Sciences Cartridge Guard E ( mml x. mm I.D.) Gradient Program : Time B conc. (%) Flow rate (ml min) Injection Volume : µl [for Steroids] : Phenomenex Kinetex 2.6u XBC8 ( mml x 2. mm I.D., 2.6µm) Flow Rate :.3 ml/min Gradient Program : Time B conc. (%) Injection Volume : µl 3

4 analysis in single FIA/LCMS/MS platform Mass (LCMS8 triple quadrupole mass spectrometry) Ionization : heated ESI Nebulizing Gas Flow : 3 L / min Heating gas flow : L/min BH Temperature : ºC MRM parameter : Drying Gas Pressure : L / min DL Temperature : 2 ºC Interface Temperature : 4 ºC Q>Q3 IS Q>Q3 Q>Q3 IS Q>Q3 Phe 66.>2. Phe IS 72.>26. C 246.2>8. C IS 2.2>8. Leu 32.>86. Leu IS 3.>89. CDC 276.>8. CDC IS 279.>8. Met.>4. Met IS 3.>7. COH 262.2>8. COH IS 26.2>8. Tyr Val 82.>36. 8.>72. Tyr IS Val IS 88.> >8. C6 C8 26.2> >8. C8 IS 29.2>8. Cit Arg 76.>3. 7.>7. Cit IS Arg IS 78.>. 8.>7. C C2 36.2> >8. C2 IS 347.3>8. Ala 9.>44. Ala IS 94.>48. C >8. C4 IS 38.3>8. C C2 62.>3. 24.>8. C IS C2 IS 7.>3. 27.>8. C6 C6OH 4.3> >8. C6 IS 43.3>8. C3 C4 C4OH 28.> > >8. C3 IS C4 IS 22.> >8. C8 C8OH 428.4> >8. C8 IS 43.4>8. R.T(min) Q>Q3 CE (V) R.T(min) Q>Q3 CE (V) Cortisol. 363.> > CortisolD2. 36.> > DOF > > DOFD > > DOF > > DOFD > > AD > > AD3C >. 29.> OHP >9. 33.> OHPD > >

5 analysis in single FIA/LCMS/MS platform DBS samples (d = mm) were placed in 96well plates, and AAs, ACs and steroids were extracted with 8 μl of acetonitrilewater solution consists of the known concentrations of stable isotope labeled standards of each compounds. The extraction were performed in an ultrasonic bath for 3 min. Samples were measured using a Nexera UHPLC system coupled to LCMS8 triple quadrupole mass spectrometer (Shimadzu Corporation, Japan). Result DBS provides a number of advantages, for examples, a less invasive and much simpler sample collection method rather than venipuncture technique. Furthermore, it provides you simpler storage and transportation as well as it can lower the infection risk of various pathogens, and requires a smaller blood volume. To date, DBSLCMS/MS has emerged as an important method for quantitative analysis of small molecules. Previously we developed an innovative AAs and ACs screening method makes it possible to inject just µl of sample and successfully reduce analytical run time as fast as 74 seconds (conventional method >2 sec.) using the combination of Nexera MP and LCMS84 (Shimadzu Corporation, Japan). In addition to that, we independently developed a method for steroids in DBS. Steroids were separated on a Phenomenex kinetex XBC8 (x2mm, 2.6μm) at a column temperature of 4 C for min. In this study, we present a strategy for performing AAs, ACs and steroids analysis within a single LC/MS/MS platform. AAs, ACs and steroids were extracted from only one dried blood spot. This system enables to automatically analyze 7 min in all target analytes in 2 injections. B conc. (%). 9 9 Injection.7 for steroids. Injection.3 for AAs, ACs min min 4 min min Analysis of AAs and ACs wash valve switching Flow rate (ml/min) equilibration Analysis of steroids wash valve switching Figure 2 Time Program of amino acids, acylcarnitines and steroids analysis

6 6 Integration of amino acid, acylcarnitine and steroids analysis in single FIA/LCMS/MS platform Table Data Summary of 8 amino acids, 7 acylcarnitines and steroids No.32 No Phe Leu Met Tyr Val Cit 4.33 Arg Ala C C C3.. C4 No.36.. C4. 7%. C4OH.. C.2 CDC.72.6 COH C6.2 3 C8.2 3 C. 2 C C6. C6OH.7.6 C8 C8OH No.322 No %.9 No.323 No No % % No % No % % No.324 No.364 target % % % 4.4

7 analysis in single FIA/LCMS/MS platform Conc. Area %RSD S/N LOD LOQ Conc. Area %RSD S/N LOD LOQ, , , , Cortisol ,97 322, AD 7 2,247,236 2,87, , ,46, ,37, ,43, , , , , DOF ,22 7, OHP 7 2 4,27 874, , , ,, ,694, , , DOF ,44 964, ,29, ,872, Conclusions This platform is an effective tool for an initial screening and able to minimize sample consumption down to ul. However, the use of a high performance LC/MS/MS system is highly recommended in order to achieve the appropriate level of sensitivity for the steroids especially. Using high performance LC/MS/MS (LCMS8, Shimadzu Corporation, Japan), the observed limit of detection (LOD) for the analysis of 7hydroxyprogesterone was.9 ng/ml. First Edition: May, 2 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 its 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 publication is based upon the information available to Shimadzu on or before the date of publication, and subject to change without notice. Shimadzu Corporation, 2