Waters Integrity System Applications

Identifying Anti-Inflammatory Drugs

Highlights: Analysis of veterinary anti-inflammatory drugs using combined photodiode array and mass spectra to confirm identity.

Photodiode array detectors produce UV spectra which can be used for identification of compounds through spectral matching. Many UV spectra are quite similar and without reference spectra cannot be used for positive compound identification. The ability of the photodiode array detector to provide information on peak homogeneity combined with the availability of commercial mass spectral libraries simplifies the identification of unknowns and increases confidence in chromatographic peak identification.

183 0.16 0.035 5.933 minutes 77 5.927 minutes 50 - 330 m/z 0.14 200 - 400 @ 1.2 nm 0.030 308 0.12

0.025 Phenylbutazone 93 0.10 238.6 Peak nm 0.020 UV Photodiode 0.08 Intensity 119 Array and EI Mass AU 0.06 0.015 252 Spec 105 data from a single 146 0.04 0.010 55 133 64 injection 160 0.02 0.005 0.00 0.000 200.00 220.00 240.00 260.00 280.00 300.00 320.00 340.00 360.00 380.00 50.00 100.00 150.00 200.00 250.00 300.00 nm m/z PDA UV Profile MS Spectrum from Response for Ion Current Phenylbutazone Peak Response for Naproxen Phenylbutazone 0.60 Peak 0.50 The combination of mass spectral and photodiode 0.40 array detection provides a Anti-Inflammatory complete picture of the 0.30 Drug Mixture chromatographic peaks in Meclofenamic 100ng sample 0.20 Acid injected on column a separation. The Ketoprofen anti-inflammatory drug Oxyphenbutazone mixture shows reasonable 0.10 Flunixin response in both the UV and the PDA at the 100 0.00 ng level injected. 0.00 2.00 4.00 6.00 8.00 10.00 12.00 Copyright 1998 Waters Corporation. Minutes 0.03 Peak #4 183 77

38.000 0.080 0.02 0.075 Purity 36.000 Intensity 308 34.000 0.070 Noise + Solvent (1.00) 32.000 0.01 93 119 0.065 252 30.000 55 105 146 0.060 64 133 28.000 0.055 0.00 26.000 0.050 Site of Max 24.000 60.00 100.00 140.00 180.00 220.00 260.00 300.00 0.045 Homogeneity 22.000 AU 0.040 20.000 Match #1 Fit 92 183 0.035 18.000 0.03

0.030 16.000

0.025 14.000 0.02 77 308 12.000 0.020 Intensity 10.000 0.015 0.01 8.000 0.010 Site of Max 105 252 Impurity 6.000 93 0.005 4.000 0.00 0.000 2.000 60.00 100.00 140.00 180.00 220.00 260.00 300.00 5.70 5.80 5.90 6.00 6.10 6.20 6.30 6.40 0.000 Minutes 0.02 Millennium PDA Purity Plot and MS Difference Triple Plot for Phenylbutazone Peak 93 119 PDA 57 77 104 133146160 189 252 Purity Angle: 0.71 Threshold: 1.05 0.00 MS Intensity Fit 92 Contamination 15 Phenylbutazone -0.02 60.00 100.00 140.00 180.00 220.00 260.00 300.00 m/z Wiley Library Reference Spectra

Serial: 48374 CAS Reg No: 129-20-4 MW: 324 Form: C19 H20 N2 O3 Oxyphenylbutazone 1-(P-HYDROXYPHENYL)-2-PHENYL-4-BUTYLPYRAZOLIDINE

199 93 324

77 N 121135 O 109 N 148162176 268 226 252 281 308 75 100 125 150 175 200 225 250 275 300 325 HO O (CH2)3 Me

Serial: 45890 CAS Reg No: 50-33-9 MW: 308 Form: C19 H20 N2 O2 Phenylbutazone 1,2-DIPHENYL-4-BUTYLPYRAZOLIDINE-3,5-DIONE 183 77 308 N O N 105 93 119 293 152 211235252 O (CH23)Me 75 100 125 150 175 200 225 250 275 300 325

Phenylbutazone and oxyphenylbutazone differ only in a hydroxyl group, but the mass spectra are clearly distinct and allow for positive compound identification.

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