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Development of HPLC-UV/MS-MS methods applied to the quality control of ursodeoxycholic acid in oral liquid pediatric formulations and raw material Pharm. Boscolo Oriana CIDEC – Tecnología Farmacéutica Facultad de Farmacia y Bioquímica Universidad de Buenos Aires

BILE ACIDS Ø steroid compounds, derived hydroxyl from 5β-24 -cholanic acid and cholesterol derivatives, synthesized in the liver. Ø components of bile that form mixed micelles with phospholipids and cholesterol. BILE ACID R 3 R 7 R 12 R 24 Cholic acid -OH -OH Chenodeoxycholic acid -OH -H Glicine (75%) Deoxycholic acid -OH -H -OH Taurine (24%) Lithocholic acid -SO 3 -2/-OH -H -H -OH (<1%) Ursodeoxycholic acid -OH -H

BILE ACIDS FUNCTION Regulate their own synthesis and enterohepatic circulation “digestive surfactants” BA s Facilitate the intestinal digestion and absorption of dietary fat, steroids, drugs, and lipophilic vitamins Signaling molecules that are involved in the regulation of lipid, glucose, and energy metabolism, drug metabolism, and immune response modulation

URSODEOXYCHOLIC ACID (UDCA) v Also known as ursodiol, is a secondary acid and the least hydrophobic of bile acids (BA). v Origin: “urso” = bear. v 7α CDCA 7β UDCA epimerase v Present in small amounts in the human beings: 1 -3 % BA

v The chemical structure determines its physicochemical properties Detergency Hydrophobicity < n˚ OH⁻ > hydrophobicity > detergency Orientation 7α > 7 β UDCA > HYDROPHILIC < TOXIC 7β 7α

PEDIATRIC APPLICATIONS LITHOLYTIC AGENT COMPLEMENTARY IN HEPATIC CHOLESTASIS DISORDERS • • Intrahepatic Cholestasis Progressive Familial Intrahepatic Cholestasis (PFIC) Alagille Syndrome Biliary Atresia UDCA help normalize biochemical and histological markers and clinical symptoms.

MECHANISMS OF ACTION UDCA Cytoprotective effects on hepatocytes and bile duct epithelial cells Dissolution of Gallstones Displacement of toxic endogenous bile acids through UDCA Anti-apoptotic effects Hepatocyte membrane protective effects

PEDIATRIC PATIENTS ORAL ADMINISTRATION OF LIQUID FORMULATIONS UDCA SUSPENSIO NS Dose adjusted to patient requirement Avoids problems associated to solid dosage forms in oral administration Very slightly soluble in water Orphan formulation

Development of an HPLC-UV analytical method for the UDCA determination in a pediatric suspension Suspension Compounds %P/V UDCA 2. 5 Glicerine 50 Vehicle q. s. 100 Sample Preparation 0. 5 g de suspension/25 m. L methanol Sonication 5‘ Centrifugation 10‘ 4000 rpm Vehicle Compounds %P/V Methylparaben 0. 08 Propylparaben 0. 02 Xanthan Gum 0. 2 Water q. s. 100 1/2 mobile phase (ACN : 0. 5 m. M H₃PO₄ p. H 3) (50: 50)

Chromatographic System ISOCRATIC ELUTION Column Symmetry C 18 ( 4. 6 mm x 150 mm i. d, 5 µm) Temperature : 40˚C Injection volume: 100 µl Wavelenght: 200 nm Flow: 1 m. L/min Mobile phase: ACN : 0. 5 m. M H₃PO₄ p. H 3 (48: 52) Run time: 8 min

Optimization and Validation Parameters SPECIFICITY LINEARITY LOD LOQ PRECISION ACCURACY ROBUSTNESS

SPECIFICITY Specificity was evaluated with different excipients that could act as potential interferences. Glicerine Xanthan gum Methylparaben Prophylparaben

Chromatograms of potential interference. A: excipient blank: (1) Methylparaben; (2) Prophylparaben. B: Sample suspension: (1) Methylparaben; (2) Prophylparaben; (3) UDCA

VALIDATION Linear range (µg/ml) 202 -606 y=216, 87 x +2744, 9 R 2 0. 9903 LOD (µg/m. L) 0. 32 LOQ (µg/m. L) 1. 08 Precision (RSD) Intraday (n=6) Response Migration time 0. 81 0. 14 Interday (n=18) Response Migration time 2. 02 0. 45 Accuracy 80% 99. 6 % (n=3 RSD= 0. 49) 100% 101. 3 % (n=3 RSD= 0. 25) 120% 99. 2 % (n= 3 RSD= 0. 23)

ROBUSTNESS Plackett Burman design Variables Column temperature Units ºC Buffer p. H Abbreviation High level Low level A, a 42 38 B, b 3, 2 2, 8 % Organic solvent % C, c 52 48 Flow m. L min-1 D, d 1, 2 0, 8 Injection mode - E, e Push Pool Wavelenght nm F, f 202 198 Sample sonication - G, g 7 3 time

SAMPLE ANALYSIS Suspension Label content (%) Content (%) 2. 5 2. 7 106. 6 % (n=3 RSD= O. 66 )

UDCA SUSPENSION CONTENT (1): UDCA Standard solution 307 µg/m. L; (2): Excipient blank; (3): UDCA suspension 265 µg/m. L

MASS SPECTROMETRY “The basis in mass spectrometry (MS) is the production of ions, that are subsequently separated or filtered according to their mass -to-charge (m/z) ratio, and detected. The resulting mass spectrum is a plot of the (relative) abundance of the produced ions as a function of the m/z ratio. ” Elucidate chemical structure MS Isotopic relationship in the samples Qualitative-quantitative content of organic and inorganic analytes in complex samples

INSTRUMENTATION Liquid Chromatography Ulimate 3000 (Thermo Fisher Scientific TSQ Quantum Access MAX (Thermo Fisher Scientific) Data System: Xcalibur 2. 1.

COMPONENTS HIGH VACUUM API - ESI TRIPLE QUADRUPOLE INJECTOR SAMPLE INTRODUCTION: • Direct Injection • Througt HPLC without chromatography separation • Througt HPLC with chromatography separation ELECTRON MULTIPLIER XCALIBUR 2. 1 MASS SPECTRUM

HPLC/MS-MS METHOD DEVELOPMENT FOR RELATED SUBSTANCES DETERMINATION IN UDCA RAW MATERIAL Bile acids may be present in UDCA raw material as related substances: LCA, which is toxic and CDCA, associated with certain secondary effects. The official USP UDCA monograph describes the determination of these related substances by thin layer cromatography (TLC). v 0. 05% LCA v 1. 5% CDCA

IDENTIFICATION OF RELATED SUBSTANCES HPLC Detectors UV Low sensitivity MASS High sensitivity § Low BA absortion in the UV-visible region (195 -201 nm) • UDCA 40 mg/m. L overload • UDCA 400 µg/m. L overload

Chromatographic System ANALYTICAL CONDITIONS- HPLC ISOCRATIC ELUTION Column Symmetry C 18 ( 4. 6 mm x 150 mm i. d, 5 µm) Temperature: 35˚C Injection volume: 25 µl Flow: 0. 4 m. L/min Mobile phase: Me. OH : ACN : 10 m. M ammonium acetate buffer p. H 7. 6 (40: 20) Analysis time: 17 min

MS CONDITIONS POLARITY Negative ANALYSIS MODE SIM

VALIDATION PARAMETERS LCA CDCA Linear range 57 – 456 (ng/m. L) 208 – 1250 (µg/m. L) Y= 415, 22 X + 14450 Y= 280492 X + 1. 10⁶ R² 0. 9883 0. 9880 LOD 0. 09 ng/m. L 3. 9 ng/m. L LOQ 0. 29 ng/m. L 13 ng/m. L PRECISION Intraday (n=6) Response 1. 69 0. 84 Migration time 0. 57 0. 40 Interday (n=12) Response 1. 85 1. 24 Migration time 1. 34 1. 08

Overload UDCA STD: 403. 2 µg/m. L – CDCA STD: 6. 65 µg/m. L – LCA STD: 228 ng/m. L

CDCA AND LCA CONTENT IN RAW MATERIAL LCA (%) CDCA (%) Limit 0. 05 1. 5 Content 0. 01 0. 2

Conclusions v An HPLC-UV method for the analysis of UDCA content in oral suspensions was developed. This method complies with ICH guidelines and is simple, fast, accurate, precise, robust and sensitive. v An HPLC-MS/MS method to quantify UDCA, CDCA and LCA was developed for the first time. It complies with USP related substances limits and validation parameters. v These methods are suitable for quality control in raw material as well as pharmaceutical formulations.

v 7 th Pharmacovigilance & Pharmaceutical Industry Conference. v Especially, Isaac Salvatore. Conference Organizer, Pharmacovigilance 2016. v. University of Buenos Aires. v. CONICET THANK YOU VERY MUCH VIELEN DANK MUCHAS GRACIAS TANTE GRAZIE

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