J. Elek , A. Simon, J. Török, A. Csontos, Cs. Ballai APPLICATION AND VALIDATION OF MID-RANGE INFRARED SPECTROSCOPY IN GLP ENVIRONMENT J. Elek , A. Simon, J. Török, A. Csontos, Cs. Ballai WSC10, Samara

Typical analytical workflow method development validation (EP, USP, ICH, etc.) analysis GLP, GMP documentation, archivation

Purpose of this presentation Why this method was made: for a certain analysis Why it was presented in Hungary: analists do not trust multivariate methods Playing around with statistics Faliure of instrument distributors „one button” solutions Dominance of HPLC and GC „counterstrike” Why it was presented Here: „János, you do not know much about chemometrics, but you play the guitar a nice way, so you should come back for the next meeting too.” Beata Walczak, 2012, Drakino

The infrared spectroscopy Infrared domain: 780 nm –1000 μm, (10 cm-1–12 500 cm-1) Discovery: Early 1800’s (Sir William Herschel) Identification of the first absorption bands: 1882-1900 Systematic examinations, Coblentz 1903 (2-3 hours per spectrum) The golden age: structure elucidation Renaissance: multivariate analysis(discriminant analysis, quanification, PCA, LDA, PLS, etc.)

Ethanol IR spectrum

Ethanol NIR spectrum

What is the goal? regression discrimination

Analitical nightmare Quantitative determination of an oily, high viscosity, mulit-component test item from arachis oil. Sensitive to hydrolysis Non volatile Normal phase chromatography??? ATR FT-IR spectroscopy Unscrambler X

ATR-technique Versatile Fast No sample preparation Easy to clean (cross-contamiation)

Absorbtion IR spectrum of the test item

TI vs peanut oil

Calibration series

Calibration series 1st derivative

PLS-DA plot

Calibration line (validated)

Validation parameters Repeatability ccal (mg/g) L4_001 5.3018 L4_002 5.2958 L4_003 5.1785 L4_004 5.3107 L4_005 5.2403 L4_006 5.2768 L4_007 5.2274 RSD% 0.9 Target C (mg/mL) cNom (mg/g) ccalc (mg/g) Recovery (%) RSD% 5 5.75 5,68 98.8 1.6 5.62 5.54 98.6 1.3 5.61 5.33 94.9 2.8 100 106.20 106.43 100.2 0.4 106.75 106.84 100.1 0.1 106.71 106.77 Date R2 RMSECV Deviation of the individual calibration points % clow chigh Feb 19 1.00 0.07 0.23 -2.4-2.6 -0.5-0.4 Feb 20. 0.04 0.24 -2.1-2.0 -1.0-0.4 Feb 24. 0.08 0.50 -3.9-3.7 -4.6-1.2 March 5 0.05 0.67 -2.6-2.4 -5.0-4.1

Achilles-heel: specificity During method development in 2.5 mg/ml solution

Regression coefficients (no derivation)

Weighted regresion coefficients

Achilles-heel: specificity

Summary The quantitative MIR analysis can replace the frequently used chromatographic techniques in most cases Minor need for sample preparation Fast, cheap With careful use of chemomertic techniqes the methods can be validated according to the mainly applied guidlines (ICH, EP, USP, etc.) Until these algorithms run in R and Matlab it will always be a very hard work to convince the „simple” analysts (and the authorities) about the adequacy of their use.