Pharmaceutical Applications
Transcription
Pharmaceutical Applications
HPLC Columns PHARMACEUTICAL APPLICATIONS A P P L I C AT I O N N O T E S Morphine, Hydromorphone and 6-MAM 1,4-Benzodiazepines in Urine LC-MS Analysis in Plasma Samples Conditions LC-MSMethod with SPE Method Conditions Method Conditions Method Conditions Method Conditions Column: Cogent Diamond Hydride 2.õ™, 2.2µm, 12 Method Conditions Catalog No.: 70200-05P-2 Column: Cogent Diamond Hydrid2 Column: Cogent Diamond Hydride Method Conditions Method Conditions Column: Cogent Column: Cogent2.1 Diamond Hydride 2.õ™,Diamond 2.2µm, 120A Hyd Dimensions: x 50 mm Catalog No.: / 0.1% 70200-05P-2 Catalog No.: Catalog No.: 70200-05P-2 formic acid (v/v) Mobile Phase: A: DI H2O70200-05P-2 Catalog No.: 70200-05P-2 Dimensions: x 50 mmacid (v/v) Dimensions: 2.1 x 50 Dimensions: 2.12.1 x0.1% 50 mm B:mm Acetonitrile/ formic Dimensions: 2.1 xH 50 mm Mobile Phase: A: DI Phase: H / (min.) 0.1% A: formic (v/v) 2OPhase: / 0.1% formic Mobile A: acid DI Gradient: time %B time (min.) %Bac 0.1% formic Mobile DI H2O 2/O B: Acetonitrile/ 0.1% formic acid (v/v) Mobile Phase: A: DI H O / 90.1% 0.1% form 2 0 85 20form B:time Acetonitrile/ 0.1% formic Gradient: time (min.) %B B: Acetonitrile/ (min.) %B 2 6 70 10 85 fo B: Acetonitrile/ Gradient: time (min.) %B 0.1% 0 85 time 9 20 Gradient: (min.) %B timt 7 20 6 70 10 8585 Gradient: time (min.) %B 0 0 85 Post Time: 7 3 min 20 06 85 70 70 PostFlow Time:Rate:3 min 0.4 mL/min 6 7 Vol.: 1 µL FlowInjection Rate: 0.4 mL/min 7 6 20 20 70 Injection Vol.: 1 µL Spiked urine sample was Sample: Extraction 3 method: Post Time: 3 min Post Time: min 7 20 Sample: Extraction method: Spiked urine sample was loaded into SPE cartridge I (Clean Screen Flow Rate: mL/min Flow Rate: 0.430.4 mL/min loaded into SPE cartridge I from (Clean Screen Post Time: min Xcel™ purchased UCT Bristol, PA, USA Injection Vol.: 1 µL Xcel™and purchased from UCT Bristol, PA, USA) Injection Vol.: µL eluted1 with 0.78 mL of acetonitrile, 200 Flow Rate: 0.4 mL/min and eluted withof0.78 mL of acetonitrile, 200ofSpiked Sample: Extraction method: Spikeu Sample: Extraction microL 2-propanol, 20method: microL ammonia Injection Vol.: 1 µL microL of 2-propanol, 20 microL of ammonia. loaded into SPE cartridge After the elution, the sample was dried unde loaded SPE cartridge I( After the elution, the sampleinto was dried under N2 Sample: Extraction method: Spik gas and dissolved inpurchased 100 50% Xcel™ purchased from U gas and dissolved in 100 microL ofmicroL 50% of Xcel™ from UCT methanol/50% DI water/0.1% formic acid. Beo loaded into SPE cartrid methanol/50% DI water/0.1% formic acid. Before and eluted with 0.78 mL and eluted with 0.78 mL of a injection, the spiked 10ppmsample spikedwas sample filter injection, the 10ppm filteredwas 20 microL of 2-propanol, Xcel™ purchased from microL of 2-propanol, mic a 0.45 μm nylon syringe filter20 (Micro throughthrough a 0.45 μm nylon syringe filter (MicroSolv After the elution, sam and eluted with 0.78 m Tech Corp). Tech Corp). After the elution, thethe sample Detection: 22-propanol, TOF mass gas and dissolved in 100 Detection:ESI – Pos ESI –– Perkin Posgas – Elmer Perkin Elmer AxION TOF m andAxION dissolved in2100 mi microL of 2 spectrometer. spectrometer.methanol/50% DI water/0 DI water/0.1% After the elution, the sa t0: t : 0.9 min0.9 min methanol/50% Column: Catalog No.: Dimensions: Mobile Phase: Cogent Diam 70200-05P-2 2 1 2.1 x 50 mm A: DI H O / 0 B: Acetonitrile 3 4 Gradient: time (min.) % 0 6 7 Post Time: 3 min Flow Rate: 0.4 mL/min Injection Vol.: 1 µL Sample: Extraction me Detection: ESI – POS - Agilent 6210 MSD TOF mass spectrometer. loaded into SP Discussion Peaks: Peaks: 1. temazepam m/z [M+H] Xcel™ purcha ¯ column was successfully used Discussion 1. temazepam301.0739 301.0739 m/z [M+H] The Cogent Bidentate C18 2.o™ 2. diazepam [M+H] 2. diazepam285.0790 285.0790 [M+H] in the analysis of an important class of drugs in plasma samples. 3. nordiazepam 271.0633 [M+H] 0 injection, the 10ppm spike 3. nordiazepam 271.0633 [M+H] eluted ¯validation injection, the 10ppm spiked Thepresented Cogent Bidentate 2.o™ columncan was 4. midazolam 326.0855 [M+H] The procedureC18 after besuccessfully used as a used gas andand dissolved inwit 10 4. midazolam 326.0855 [M+H] through a 0.45 μm nylon in the analysis analysisof ofplasma an important class of drugs in plasma Discussion routine samples (or whole blood samplessamples. – through a 0.45 μm nylon syr microL DI of wate 2-pr Discussion methanol/50% after theprocedure extraction procedure) for thecan presence of as a Thechanging presented after validation be used Tech Corp). Tech Corp). morphine, hydromorphone, or 6-MAM (indicator of heroin routine analysis of plasma samples (or whole blooduse). samples – The Cogent Diamond Hydride 2.õ™ column was successfully usedelutio After the injection, the 10ppm spu Detection: ESI – Pos – Perkin Elme The Cogent Diamond Hydride column was successfully Peaks: Detection: ESI – Pos – Perkin Elmer A in analysis of 1,4-benzodiazepines in2.õ™ urine samples after SPE after changing the extraction procedure) for the presence of Peaks: APP A-310 APP A-305 through a 0.45 nylo spectrometer. 1. temazepam 301.0739 m/z [M+H] in analysis of 1,4-benzodiazepines in urine samples after gas andμm disso extraction. Four available compounds were well retained and morphine, hydromorphone, or 6-MAM (indicator of heroin use). 1. temazepam 301.0739 m/z [M+H] spectrometer. For more information For more information 2. diazepam 285.0790 [M+H] extraction. Four available compounds were well retained separated. The procedure could be used for determination of this 0.9 min Tech Corp). 2. diazepam 285.0790 [M+H] 0: t0: tThe 0.9 min methanol/50% 3. nordiazepam 271.0633 [M+H] classseparated. of compounds inprocedure urine samples andbe other body could used forfluids. determination of www.MTC-USA.com or www.MTC-USA.com or 3. nordiazepam 271.0633 [M+H] 4. midazolam 326.0855 [M+H] Detection: ESI –and Pos – Perkin El 4. midazolam 326.0855 [M+H] Peaks: other body fluids. class of compounds in urine samples injection, the 1 [email protected] [email protected] Discussion spectrometer. 1. temazepam 301.0739 m/z [M+H] Discussion through a 0.45 2. diazepam 285.0790 [M+H] t0: 0.9 min 3. nordiazepam 271.0633 [M+H] The Cogent Diamond Hydride 2.õ™ column Tech Corp). The Cogent Diamond Hydride 2.õ™ column w 4. midazolam 326.0855 [M+H] in analysis of 1,4-benzodiazepines in uri Detection: ESI – Pos –P in analysis of 1,4-benzodiazepines in urine Discussion Peaks: extraction. Four available compounds w extraction. Four available spectrometer compounds were Acetaminophen Impurities Method 1. temazepam 301.0739 m/zCough Syrup Ingredients [M+H] + separated. The procedure could be used f separated. The procedure could be used for 2. diazepam 285.0790 [M+H] + The Diamond 2.õ™ and coluo class of compounds inHydride urine tCogent 0.9samples min 0:compounds Robust and Easy APAP Method Separation of+ Antitussives, Analgesics, Decongestants class of in urine samples and oth 3. nordiazepam 271.0633 [M+H] ¯ Column: Cogent Bidentate 2.2µm, ¯ 2.o™, Column: Cogent Bidentate C18 C18 2.o™, 2.2µm, 120A 120A Catalog No.: 40218-05P-2 40218-05P-2 Catalog No.: Dimensions: 2.12.1 50 mm Dimensions: x 50x mm Mobile Phase: A: DI 0.1% formic acid (v/v) / 0.1% formic acid (v/v) Mobile Phase: A: H DI2OH/2O B: 50% Acetonitrile / 50% Methanol / B: 50% Acetonitrile / 50% Methanol / 0.1% formic acid (v/v) 0.1% formic acid (v/v) Gradient: time (min.) %B Gradient: time (min.) %B 0 0 5 5 4 50 4 50 5 90 5 90 6 90 7 6 5 90 7 5 Post Time: 3 min PostRate: Time: 0.43mL/min min Flow Flow Rate: 0.4 mL/min Injection Vol.: 1 µL Peaks: 286.1438 m/z [M+H] + Injection Vol.:1. Morphine 1 µL ++ 2. Hydromorphone 286.1438 m/z[M+H] [M+H] Peaks: 1. Morphine 286.1438 m/z 3. 6-Monoacetylmorphine (6-MAM)m/z [M+H] + 2. Hydromorphone 286.1438 + 328.1543 m/z [M+H] 3. 6-Monoacetylmorphine (6-MAM) Detection: ESI328.1543 – POS - Agilent 6210 MSD TOF mass + m/z [M+H] spectrometer. + + + + + + + + + + + + + + + + + + + + 4. midazolam 326.0855 [M+H] + class ofCogent compounds in urine samples an Phenyl Hydride™ 4µm, 100Å Column: Column: CatalogNo.: No.: Catalog Dimensions: Dimensions: MobilePhase: Phase: Mobile Column: Cogent Cogent Bidentate C18™, 4µm, 100A Column: Bidentate C18™, 4µm, 100A Catalog 40018-75P Catalog No.:No.:40018-75P Dimensions: 75 mm Dimensions: 4.6 x4.6 75xmm Solvents: A: DIA:HDI H2O/ 0.1% Solvents: formicformic acid acid 2O/ 0.1% B: Acetonitrile/ B: Acetonitrile/ 0.1% 0.1% formicformic acid acid Gradient: (min.) %B %B time (min.) %B Gradient: timetime (min.) time (min.) %B 0 0 0 6 0 6 30 30 1 1 0 6.01 6.0110 0 10 4 4 30 30 10 1010 10 Post Time: Post Time: 3 min 3 min Injection Vol.: 5 microL Injection Vol.: 5 microL Flow Rate: 1.0 mL/min Flow Rate: UV 280 1.0 mL/min Detection: (4-aminophenol, acetaminophen) and Detection: 324 UV (4-aminophenol, acetaminophen) and nm 280 (4-nitrophenol) 324 nm (4-nitrophenol) Peaks: 1. 4-aminophenol 1.072 min Peaks: 1. 4-aminophenol 2. acetaminophen 4.6681.072 min min 2. acetaminophen 4.668 min 3. 4-nitrophenol 7.588 min t0: 0.9 min 3. 4-nitrophenol 7.588 min t0: Gradient: Gradient: Cogent Phenyl Hydride™ 4µm, 100Å The Cogent Diamond Hydride 2 69020-7.5P 69020-7.5P x 75 x 75 mmmm of 1,4-benzodiazep in4.64.6 analysis H /O0.1% / 0.1% A: A: DI DI HO TFATFA (v/v)(v/v) Acetonitrile/ 0.1%TFA (v/v) extraction. Four available com B: B: Acetonitrile/ 0.1%TFA (v/v) time (min.)%B%B time (min.) separated. The procedure coul 0 5 0 5 5 2 2 class of5 compounds in urine sa 11 50 Post PostTime: Time: Flow FlowRate: Rate: Injection Vol.: Injection Vol.: Sample: Sample: 0.9 min Detection: Detection: t0: t0: Discussion Acetominophen and two of its major impurities were analyzed using the Cogent Bidentate C18™ column and a simple mobile Acetominophen and two of its major impurities were analyzed phase. The peak shapes were very high. The repeatability of the Cogent Bidentate C18™ =column theusing results was extremely good (%RSD 0.01). and a simple mobile phase. The peak shapes were very high. The repeatability of the results was extremely good (%RSD = 0.01). Peaks APP A-249 For more information www.MTC-USA.com or [email protected] Discussion procedure could be used Method separated. Conditions The Method Conditions Method Conditions Method Conditions Discussion in analysis of 1,4-benzodiazepines in u and Preservatives extraction. Four available compounds 1. Acetaminophen 2. Pseudoephedrine 3. Guaifenesin Discussion Discussion 4. Benzoic Acid 5. Methyl Paraben 6. Dextromethorphan 7. Propyl paraben APP A-174 For more information www.MTC-USA.com or [email protected] www.MTC-USA.com 2 2 11 50 1212 5 5 3 min 3 min 1.01.0 mL/min mL/min 2 µL 2 µL Stock Solution: 1 mg/mL solutions of each Stock Solution: 1 mg/mL solutions of each analyte were made using a 50/50 solvent analyte were made using a 50/50 solvent A/solvent B diluent (v/v). A/solvent B diluent (v/v). Working Solution: 0.1 0.1 mg/mL dilutions werewere Working Solution: mg/mL dilutions made of the stock solutions and and usedused for peak made of the stock solutions for peak identity confirmations. A 0.1 mg/mL mixture of identity confirmations. A 0.1 mg/mL mixture of all the analytes was also made from the stock all the analytes was also made from the stock solutions. solutions. UV 210 nm (0-6 min), 230 nm (6-15 min) UV 210 nm (0-6 min), 230 nm (6-15 min) 0.9 min 0.9 min Cold and cough formulations may contain a number of components Cold and formulations may contain a number of components such as cough antitussives (dextromethorphan), decongestants such as antitussives decongestants (pseudoephedrine, guaifenesin),(dextromethorphan), analgesics (acetaminophen), and (pseudoephedrine, guaifenesin), analgesics (acetaminophen), preservatives (methyl paraben, propyl paraben, benzoic acid). The and preservatives paraben, paraben, benzoic acid). method illustrates(methyl not only excellentpropyl separation between a variety of The these compounds, symmetric peak between shapes can be of method illustrates but not also only that excellent separation a variety obtained in each case. in particular is often these compounds, but Dextromethorphan also that symmetric peak shapes can be problematic tailingDextromethorphan due to the tertiary amine. The method obtained in terms each ofcase. in particular is often isproblematic also very reproducible, the five demonstrates. in terms ofas tailing duerun to overlay the tertiary amine. The method is also very reproducible, as the five run overlay demonstrates. HPLC Columns A P P L I C AT I O N N O T E S Oxybenzone and Octinoxate Assay Method for Levothyroxine Separation of Two APIs in Chap Stick® Extract Superior Resolution, Reproducibility & Peak Shape Method Conditions Method Conditions Column: Fig. A: MTC Phenyl Hydride™, 4µm, 100A Column: Fig. A: MTC Phenyl Hydride™, 4µm, 100A Type B silica-based Cyano, Fig. B:Fig. TypeB: B silica-based Cyano, 5µm, 100A 5µm, 100A Catalog A: 69020-7.5P Fig. B: N/A Catalog No.: No.: Fig. A:Fig. 69020-7.5P Fig. B: N/A Dimensions: 4.6 xA:754.6 mmx 75 Fig.mm B: 4.6 x 250 Dimensions: Fig. A:Fig. Fig. B: mm 4.6 x 250 mm Mobile Phase: Fig. A:Fig. A: DIA:water/ formic acidformic acid Mobile Phase: A: DI0.1% water/ 0.1% B: 97% B: Acetonitrile/ 3% DI water/ 97% Acetonitrile/ 3% DI water/ 0.1% formic acid 0.1% formic acid Fig. B: 60% DI water/ 40% acetonitrile/ Fig. B: phosphoric 60% DI water/ 0.05% acid 40% acetonitrile/ 0.05% acid Gradient: Fig. A time (min.) phosphoric %B Gradient: Fig. A 0 time (min.) %B 20 6 0 50 20 7 6 20 50 Temperature: Fig. A: 35 °C Fig. B:7 ambient 20 Flow rate: Fig. A: 1.0 mL/min Fig. B: 1.5 mL/min Temperature: Fig. A: 35 °C Fig. B: ambient Injection volume: Fig. A: 2 µL Fig. B:100 µL Flow rate: Mix of levothyroxine Fig. A: 1.0and mL/min Fig.standards B: 1.5 mL/min Sample: liothyronine Injection volume: Fig. 0.4 A:mg2levothyroxine µL Fig. B:100 Stock Solutions: or liothyronine dissolved µL with 1 mLMix 10 mM in 50:50 DI water: methanol. Working standards Sample: ofNaOH levothyroxine and liothyronine Peaks: Peaks: Discussion Method Conditions Column: Catalog No.: Dimensions: Solvents: Cogent Bidentate C18™, 4µm, 100A 40018-75P 4.6 x 75 mm A: DI H2O/ 0.1% formic acid (v/v) B: Acetonitrile/ 0.1% formic acid (v/v) Gradient: time (min.) %B 0 60 1 60 4 100 6 100 7 60 Post Time: 3 min. Temperature: 40°C Injection Vol.: 2 microL Flow Rate: 1.0 mL/min Detection: UV 288 nm Peaks: 1. Oxybenzone 2. Octinoxate t0: 0.9 min Solution (Fig. A): Aliquots of stock solutions were mixed and Stock 0.4concentrations mg levothyroxine or liothyronine dissolved diluted with 50:50Solutions: A:B to obtain of 40 mg/L and 4 mg/L for levothyroxine and liothyronine respectively. with 1 mL 10 mM NaOH in 50:50 DI Working water: methanol. Working Solution (Fig. B): Aliquots of stock solutions were solutions mixed andwere mixed and Solution (Fig. A): Aliquots of stock diluted with the mobile phase to obtain concentrations of 10 mg/L diluted with 50:50 A:B to obtain concentrations of 40 mg/L and 4 and 0.2 mg/L for levothyroxine and liothyronine respectively. mg/L for levothyroxine and liothyronine respectively. Working 1. liothyronine sodium Solution (Fig. B): Aliquots of stock solutions were mixed and 2. levothyroxine sodium diluted with the mobile phase to obtain concentrations of 10 mg/L Discussion and 0.2 mg/L for levothyroxine and liothyronine respectively. 1. liothyronine sodium 2. levothyroxine sodium The USP assay method for levothyroxine requires that a resolution of APP A-128 For more information www.MTC-USA.com or [email protected] not less than 5.0 must be demonstrated between levothyroxine and related compound liothyronine. A chromatogram obtained from Discussion following the USP method using a Type-B silica based L10 column is shown Figure B. method The average resolution between TheinUSP assay for levothyroxine requiresthe thattwo a resolution of compounds five5.0 runs is 2.8, does not satisfy the system APP A-216 not lessover than must be which demonstrated between levothyroxine and suitability for resolution for this assay. Figure A shows the five-run related compound liothyronine. A chromatogram obtained fromFor more information overlay obtained from a method developed with the Cogent Phenyl following the USP method using a Type-B silica based L10 column is HydrideTM column. The average resolution in this case was 5.3. In www.MTC-USA.com or shown in Figure B. The averagewere resolution the two addition, the peak shapes and reproducibility far superiorbetween for the compounds five runs is 2.8, which does not satisfy the system [email protected] Phenyl HydrideTM over method. This method shows how two common ingredients found in sunscreens and lip balms can be separated using the Bidentate C18™ column. The two compounds are very hydrophobic, so a mobile phase gradient with significant organic content was used in order to avoid excessive retention. Likewise, a highly organic diluent should be used to adequately extract the compounds from the lip balm material. The figure shows an overlay of two runs from different column lots, demonstrating the lot-to-lot reproducibility of the Bidentate C18™ stationary phase. suitability for resolution for this assay. Figure A shows the five-run overlay obtained from a method developed with the Cogent Phenyl HydrideTM column. The average resolution in this case was 5.3. In addition, the peak shapes and reproducibility were far superior for the Phenyl HydrideTM method. Atorvastatin Method Transfer Use of Near-UHPLC 2.2µm Column Alprazolam (Xanax®) Robust Separation of API from USP Internal Standard Method Conditions Method Conditions Method Conditions ¯ C18 ¯ 120A Fig. A:Fig. Cogent Bidentate C18 2.o™, 2.2um, A: Cogent Bidentate 2.o™, 2.2um, 120A Fig. B: Cogent Bidentate C18™, 4um, 100A Fig. B: Cogent Bidentate C18™, 4um, 100A Catalog No.: Fig. A: 40218-05P-2; Fig. B: 40018-05P-2 Catalog No.: Fig. A: 40218-05P-2; Fig. B: 40018-05P-2 Dimensions: 2.1 x 50 mm Dimensions: 2.1 x 50 mm Mobile Phase: A: DI H2O / 10 mM ammonium acetate Mobile Phase: A: acetonitrile DI H2O / 10 mMDI ammonium acetate B: 90% / 10% water / B: ammonium 90% acetonitrile 10 mM acetate/ 10% DI water / 10 mM ammonium acetate Gradient: time (min.) %B time (min.) %B Gradient: %B (min.) %B 0 time (min.) 40 6 time100 1 7 0 100 40 640 100 Flow Rate: 0.3 mL/min 1 100 7 40 Detection: UV 248 nm Flow Rate: 0.3 mL/min Sample: 40 mg strength Lipitor® tablet was ground and Detection: UV 248 nm added to a 50 mL volumetric flask with a por® tabletwas was ground and Sample: 40solvent mg strength Lipitor tion of B diluent. The solution added a 50 volumetric flasksolwith a porsonicated 10to min andmL diluted to mark with tion of solvent B diluent. The solution vent B. It was then filtered through a 0.45 µm was min and dilutedCorp. to mark with solnylonsonicated membrane10 (MicroSolv Technology Eatontown, The filtrate was diluted 4xa 0.45 µm vent NJ, B. USA). It was then filtered through in a diluent 50/50 solvent B/ 1N HCl. It was Corp. nylon of membrane (MicroSolv Technology heated in a dry bath for 10The min filtrate at 85°C.was diluted 4x Eatontown, NJ, USA). Peaks: 1. Atorvastatin in a diluent of 50/50 solvent B/ 1N HCl. It was 2. Atorvastatin lactone Method Conditions Column: Column: Peaks: Discussion APP A-308 For more information www.MTC-USA.com or [email protected] Peak 1 Peak 1 Peak 2 Peak 2 heated in a dry bath for 10 min at 85°C. 1. Atorvastatin 2. Atorvastatin lactone This application note demonstrates how data obtained on a 4um Bidentate C18™ column (Fig. B) can be adapted for a 2.2um Discussion phase (Fig. A). The retention times of both analytes are very comparable. The method excellent of the on a 4um This application noteproduces demonstrates howseparation data obtained API and its main acid degradant. It is also LC-MS compatible Bidentate C18™ column (Fig. B) can be adapted for a 2.2um and so could be used in clinical applications involving plasma phase (Fig. A). The retention times of both analytes are very samples. APP A-183 For more information comparable. The method produces excellent separation of the API and its main acid degradant. It is also LC-MS compatiblewww.MTC-USA.com or [email protected] and so could be used in clinical applications involving plasma samples. www.MTC-USA.com Column: Cogent Cogent Column: DiamondDiamond Hydride™,Hydride™, 4µm, 100A 4µm, 100A Catalog 70000-7.5P Catalog No.: No.: 70000-7.5P Dimensions: Dimensions: 4.6 x 754.6 mmx 75 mm Solvent: O/ DI 0.1% formic acidformic (v/v) acid (v/v) Solvent: A: DI H2A: H2O/ 0.1% B: Acetonitrile/ 0.1% formic acidformic (v/v) acid (v/v) B: Acetonitrile/ 0.1% Gradient: time (min.) %B Gradient: time (min.) %B 0 95 0 95 95 1 1 50 95 6 6 95 50 7 7 95 Post Time: 3 min injection 1 µL 3 min PostVol.: Time: Flowinjection Rate: 1.0 mL/min Vol.: 1 µL Peaks: 1. Triazolam (internal standard) Flow Rate: 1.0 mL/min 2. Alprazolam (API) Peaks: 1. Triazolam (internal standard) 3, 4. Impurities Detection: 254 nm2. Alprazolam (API) t0: 0.9 min3, 4. Impurities Detection: t0: Discussion 254 nm 0.9 min The USP assay method for Alprazolam uses a bare silica Discussion column and a complex mobile phase consisting of acetonitrile, chloroform, butyl alcohol, and acetic acid. In this method a The USP assay method for Alprazolam uses a bare simple LC-MS compatible mobile phase is used and produces column a complex of aceto excellent peakand shapes for both mobile the APIphase and itsconsisting USP internal chloroform, butyl alcohol, and acetic acid. In this me standard. Furthermore, a resolution of 4.3 mobile was obtained simple LC-MS compatible phasebetween is usedthe and pro two peaks, whichpeak meetsshapes the USPfor system Rs>its 2.0.USP i excellent both suitability the API ofand Two impurity peaks are also observed, which further illustrates standard. the resolution capabilities of the column. Furthermore, a resolution of 4.3 was obtained betwe two peaks, which meets the USP system suitability of R Two impurity peaks are also observed, which further illu the resolution capabilities of the column. HPLC Columns A P P L I C AT I O N N O T E S Vardenafil (Levitra®) LC-MS Compatible Assay Method Cefpodoxime Proxetil USP Assay Method Method Conditions Method Conditions Method Conditions Method Conditions Column: Cogent 4µm, 100A Column: Cogent UDA™, 4µm,UDA™, 100A Catalog No.: 40031-7.5P Catalog No.: 40031-7.5P Dimensions: 4.6 x 75 mm Dimensions: 4.6 x 75 mm Solvents: A: DI H2O / 0.1% formic acid (v/v) Solvents: A: DI H2O / 0.1% formic acid (v/v) B: Acetonitrile/ 0.1% formic acid (v/v) Acetonitrile/ 0.1%%B formic acid (v/v) Gradient: time (min.)B: %B time (min.) Gradient:0 time 90 (min.) 6 %B 40time (min.) %B 1 90 790 90 0 6 40 Post Time: 3 min 1 90 7 90 Injection Vol.: 2 microL Post Time: Flow rate: 1.0 mL/min3 min Injection Detection: UVVol.: 210 nm2 microL Sample: 20mg strength Flow rate: 1.0 Levitra® mL/mintablet was ground and addedUV to a 210 25mLnm volumetric flask. A Detection: portion of 50/50 solvent A/solvent B diluent Sample: tablet was ground was added20mg and thestrength flask was Levitra® sonicated 10 tomark a 25mL volumetric flask. A min. Then itand wasadded diluted to and mixed. A portion wasportion filtered through a 0.45 μm nylon of 50/50 solvent A/solvent B diluent syringe filter (MicroSolv Tech Corp). was added and the flask was sonicated 10 Peak: 1. Vardenafil min. Then it was diluted to mark and mixed. A t0: 0.7 min portion was filtered through a 0.45 μm nylon syringe filter (MicroSolv Tech Corp). Discussion Peak: 1. Vardenafil Vardenafil in a tablet formulation can be readily assayed with t0: compatible gradient 0.7 min Overlay of five runs this LC-MS method. The peak tailing factor APP A-266 For more information www.MTC-USA.com or [email protected] was close to unity. The compound has several amine groups which can produce tailing with ordinary HPLC columns that Discussion have a number of surface silanols. The MS-compatible mobile APPofA-149 Overlay five runs phase means that the method can be adapted to more complex Vardenafil in a tablet formulation can be readily assayed Forwith more information samples such as plasma. Five runs are shown in the figure to this LC-MS compatible gradient method. The peak tailing factor illustrate the repeatability of the data. www.MTC-USA.com or was close to unity. The compound has several amine groups [email protected] which can produce tailing with ordinary HPLC columns that have a number of surface silanols. The MS-compatible mobile phase means that the method can be adapted to more complex samples such as plasma. Five runs are shown in the figure to illustrate the repeatability of the data. Forced Degradation of Clopidogrel Separation of API and Degradants Method Conditions Method Method Method Conditions Conditions Conditions Method Conditions APP A-258 For more information www.MTC-USA.com or [email protected] Discussion t: 0 2. Cefpodoxime Proxetil, R-epimer 1.9 min The USP assay method for cefpodoxime proxetil specifies a resolution of not less than 2.5 must be obtained between the two epimers of the Discussion prodrug. Following the method using a Cogent Bidentate C18™ column, the average resolution was calculated to be 2.8. In addition, The USP assay method for cefpodoxime proxetil specifies a resol the R epimer tailing factor must be not more than 1.5. Again, this data of not than 2.5with must obtained between the two epimers o meets thisless requirement a be tailing factor of 1.2. Finally, the data prodrug. Following with the a method using a Cogent C shows good repeatability retention time %RSD from five Bidentate runs of column, the average resolution was calculated to be 2.8. In add 0.2%. the R epimer tailing factor must be not more than 1.5. Again, this meets this requirement with a tailing factor of 1.2. Finally, the shows good repeatability with a retention time %RSD from five ru 0.2%. Hormone Replacement Capsule Separation of Estriol, Estradiol and Progesterone Column: Cogent Diamond Hydride™, 4µm, 100A Column: Column: Column: Cogent Cogent Cogent Cogent Diamond Diamond Diamond Hydride™, Hydride™, Hydride™, 4µm, 4µm, 4µm, 100A 100A 100A Column: Diamond Hydride™, 4µm, 100A Catalog No.: 70000-15P-2 Catalog Catalog Catalog No.: No.: No.: 70000-15P-2 70000-15P-2 70000-15P-2 Catalog No.: 70000-15P-2 Dimensions: x 2.1 150 mm Dimensions: 2.12.1 x 150 mm Dimensions: Dimensions: Dimensions: 2.1 2.1 x x150 x150 150 mm mm mm Solvents: A: DI H / 0.1% formic acid (v/v) 2H Solvents: O /O0.1% acid (v/v) 2A: /2O 0.1% /formic 0.1% / 0.1% formic formic formic acid acid acid (v/v) (v/v) (v/v) Solvents: Solvents: Solvents:A: DIA:H A: DI DI DI HOH 2 2O Acetonitrile/ 0.1% formic acid (v/v) B:B: Acetonitrile/ 0.1% formic acid (v/v) B:B: Acetonitrile/ B:Acetonitrile/ Acetonitrile/ 0.1% 0.1% 0.1% formic formic formic acid acid acid (v/v) (v/v) (v/v) Gradient: time (min.)%B%B time (min.) Gradient: time (min.) time (min.) %B %B Gradient: Gradient: Gradient: time time time (min.) (min.) (min.) %B %B %B time time time (min.) (min.) (min.) %B %B %B 0 0 95 95 7 7 60 60 0 0 0 95 95959595 8 8 795 7 795 606060 2 2 2 22 959595 8 88 959595 Temperature: 2525 Temperature: °C°C Temperature: Temperature: Temperature: 25°C 25°C°C Post Time: 3 min 3 25 min Post Time: Post Post Post Time: Time: Time: 3min 3min min Injection Vol.: Injection Vol.:1 microL 1 3microL Flow Rate: 0.40.4 mL/min Flow Rate:Vol.: mL/min Injection Injection Injection Vol.: Vol.: 1 1microL 1microL microL Detection: ESI – POS -mL/min Agilent 6210 MSD TOFTOF mass 6210 MSD mass Detection: ESI – POS - Agilent Flow Flow Flow Rate: Rate: Rate: 0.4 0.4 0.4 mL/min mL/min spectrometer. spectrometer. 6210 6210 6210 MSD MSD MSD TOF TOF TOF mass mass mass Detection: Detection: Detection: ESI ESI ESI – –POS –POS POS - Agilent - Agilent - Agilent + Peaks: 1. 1. API: Clopidogrel, m/zm/z 322.0663 [M+H] Peaks: API: Clopidogrel, 322.0663 [M+H]+ spectrometer. spectrometer. spectrometer. 2. 2. Degradant: Clopidogrel Acid, m/z 308.0507 Clopidogrel Acid, m/z 308.0507 + + + Peaks: Peaks: Peaks: 1.Degradant: 1. API: 1.API: Clopidogrel, Clopidogrel, Clopidogrel, m/z m/z m/z 322.0663 322.0663 322.0663 [M+H] [M+H] [M+H] + API: + [M+H] 2.[M+H] 2. Degradant: 2.Degradant: Degradant: Clopidogrel Clopidogrel Clopidogrel Acid, Acid, Acid, m/z m/z m/z 308.0507 308.0507 308.0507 3. 3. Degradant: Clopidogrel N-oxide, m/z Degradant: Clopidogrel N-oxide, m/z + ++ + [M+H] [M+H] [M+H] 338.02 [M+H] 338.02 [M+H]+ 3. 3. Degradant: 3.Degradant: Degradant: Clopidogrel Clopidogrel Clopidogrel N-oxide, N-oxide, N-oxide, m/z m/z m/z t0t: : 0.90.9 min min 0 + + + Discussion t0:t0t:0: Discussion Column: Cogent Bidentate 4µm, 100Å Column: Cogent Bidentate C18™, C18™, 4µm, 100Å Catalog 40018-25P Catalog No.:No.:40018-25P Dimensions:4.6 x4.6 250 mm Dimensions: 250xmm Mobile Phase: 60% 60% 20 mM acetate/ acetate/ Mobile Phase: 20ammonium mM ammonium 40% 40% acetonitrile acetonitrile Temperature: 30 °C Temperature: 30 °C Flow rate: 1.0 mL/min Flow rate: 1.0 mL/min Injection Vol.: 20 µL Injection Vol.: 20 nm µL Detection: UV 235 Detection: UV 235 nm Sample: Stock Solution: A 200 mg strength Sample: Stock Solution: A 200 strength cefpodoxime proxetil tablet wasmg ground and added to a 100 mL volumetric flask. was The ground a cefpodoxime proxetil tablet flask added was diluted mark the mobileflask. The to ato100 mLwith volumetric phase and was sonicated. A portion was then flask diluted to mark with the mobile filtered with a 0.45 micron nylon syringe phase and sonicated. A portion was then filter (MicroSolv Tech Corp). filtered with 100 a 0.45 nylon syringe Working Solution: µL ofmicron the stock filter solution was(MicroSolv diluted Tech with Corp). 900 µL of the Working mobile phase. Solution: 100 µL of the stock Peaks: 1. Cefpodoxime Proxetil, S-epimer solution was diluted with 900 µL of the 2. Cefpodoxime Proxetil, R-epimer mobile phase. t0: Peaks: 1.9 min 1. Cefpodoxime Proxetil, S-epimer 338.02 338.02 338.02 [M+H] [M+H] [M+H] 0.9 0.9 0.9 min min min A sensitive, selective, and rapid ANP HPLC–MS method was A sensitive, selective, and rapid ANP HPLC–MS method was Discussion Discussion Discussion developed for the simultaneous quantification of clopidogrel developed simultaneous quantification of and clopidogrel (Plavix®) andfor its the degradants. Separation of the drug the (Plavix®) and its degradants. Separation ofHPLC–MS the drug method and the was A A sensitive, A sensitive, sensitive, selective, selective, selective, and and rapid rapid rapid ANP ANP ANP HPLC–MS HPLC–MS method method was was degradation products underand stress conditions was successfully degradation products under stress conditions was developed developed developed forforfor the the the simultaneous simultaneous simultaneous quantification quantification quantification ofofclopidogrel ofclopidogrel clopidogrel achieved on a Cogent Diamond Hydride™ column. Thesuccessfully method achieved onand aand Cogent Diamond Hydride™ column. The method (Plavix®) (Plavix®) (Plavix®) and itsits degradants. itsdegradants. degradants. Separation Separation Separation ofofthe of the the drug drug drug and and and the the the was transferred from UV-HPLC method. The developed method was transferred from UV-HPLC method. The developed method can be used for products the determination ofstress clopidogrel in commercial under under under stress stress conditions conditions conditions was was was successfully successfully successfully degradation degradation degradation products products can be used for the determination of clopidogrel in commercial tablets for quality control with an application to a content uniforachieved achieved achieved onon aona Cogent aCogent Cogent Diamond Diamond Diamond Hydride™ Hydride™ Hydride™ column. column. column. The The The method method method APP A-175 tablets for quality control with an application to aitcontent uniformity test. The method isfrom also stability-indicating as isdeveloped suitable was was was transferred transferred transferred from from UV-HPLC UV-HPLC UV-HPLC method. method. method. The The The developed developed method method method mity The method is also stability-indicating asits it degrais suitable For more information for the test. determination of clopidogrel in the presence of can can can bebeused beused used forforthe forthe the determination determination determination ofofclopidogrel ofclopidogrel clopidogrel inincommercial incommercial commercial its degrafor theproducts determination of clopidogrel in the presence dation under all stress conditions using HCl,ofNaOH, www.MTC-USA.com or tablets tablets tablets forfor quality forquality quality control control control with with with an an application anapplication application toto aHCl, toa content acontent content uniforuniforunifordation all stress conditions using NaOH, light and products hydrogenunder peroxide. mity mity mity test. test. test. The The The method method method is isalso isalso also stability-indicating stability-indicating stability-indicating asasitasitisitissuitable issuitable suitable [email protected] light and hydrogen peroxide. presence presence presence ofof its ofits degraitsdegradegraforfor the forthe the determination determination determination ofof clopidogrel ofclopidogrel clopidogrel inin the inthe the dation dation dation products products products under under under allallstress allstress stress conditions conditions conditions using using using HCl, HCl, HCl, NaOH, NaOH, NaOH, light light light and and and hydrogen hydrogen hydrogen peroxide. peroxide. peroxide. www.MTC-USA.com Method Conditions MethodConditions Conditions MethodMethod Conditions Column: Cogent UDC Cholesterol™ 4µm Column: Cogent UDC Cholesterol™ 4µm, 100A Column: Cogent UDC Cholesterol™ Column: Cogent UDC Cholesterol™ Catalog No.:Catalog 69069-7.5P Catalog No.: 69069-7.5P No.: 69069-7.5P Catalog No.: 69069-7.5P Dimensions: 4.6 x 75 mm Dimensions: 4.64.6 x4.6 75 Dimensions: xmm 75 mm Dimensions: x acid 75 mm Solvents: A: DI H2O/ 0.1% formic (v/v) O/ 0.1% formic acid (v/v) Solvents: A:0.1% DI Hformic 0.1% formic acid Solvents: B: Acetonitrile/ acid (v/v) O/ 0.1% formic acid (v Solvents: A:A: DI H2H 2DI 2O/ Gradient: time (min.) B: %B Acetonitrile/ 0.1% formic acid (v B: Acetonitrile/ 0.1% formic a B: Acetonitrile/ 0.1% formic aci 0 33 Gradient: time (min.) %B%B Gradient: time (min.) %B Gradient: time (min.) 2 33 33 3333 11 65 0 0 0 12 33 2 2 2 33 3333 Post Time: 3 min 11 1111 65 6565 Flow rate: 1.0 mL/min 12 1212 33 3333 Detection: UV 210 nm Injection Vol.: 10 Time: µL Post Time: 3 min Post min Post Time: 33 min Peaks: 1. Estriol Flow rate: mL/min Flow rate: 1.01.0 1.0 mL/min Flow mL/min 2.rate: Estradiol Detection: UVUV 210 nm 3. Progesterone Detection: UV 210 nm Detection: 210 nm Injection Vol.: 10 10 µL Injection Vol.: 10 Injection Vol.: µLµL Peaks: 1. Estriol Peaks: Estriol Peaks: 1.1. Estriol This gradient method features a Estradiol separation of the three 2. Estradiol 2.2. Estradiol components of a hormone replacement formulation. Excellent 3. Progesterone Progesterone 3. Progesterone separation is obtained between the3. three compounds using the Discussion Cogent UDC-Cholesterol™ column. Figure A shows a five run overlay of the formulation extract Discussion Discussion injections,Discussion demonstrating the excellent run-to-run repeatability of the method. This gradient method features a aseparation This gradient method features aand separ gradient method separat Figure This B shows a zoomed-in view so features that the estriol components of present a of hormone formul components replacement fo estradiol peaks, which are inhormone much replacement lower concentration components of a ahormone replacement form than progesterone, can beis seen clearly. Figure B also shows separation is obtained between the three compo separation obtained between the three c separation is obtained between the three com separation of an impurity from the progesterone peak. Cogent UDC-Cholesterol™ column. Cogent UDC-Cholesterol™ column. Cogent UDC-Cholesterol™ column. Figure A shows a five run overlay of of the form Figure shows fiverun runoverlay overlay ofthe thef Figure A Ashows a afive injections, demonstrating thethe excellent run-to-r injections, demonstrating theexcellent excellent run injections, demonstrating run-t of the method. the method. ofof the method. Figure B shows a zoomed-in view so so that Figure shows zoomed-in view soth Figure B Bshows a azoomed-in view estradiol peaks, which areare present in much lowe estradiol peaks, which are present much estradiol peaks, which present in in much lo than progesterone, cancan be seen clearly. Figure than progesterone, canbe beseen seen clearly. F than progesterone, clearly. Fig separation of an impurity from thethe progesterone separation an impurity from the progeste separation ofof an impurity from progester HPLC Columns TM CogentTM TYPE-C silica LC phases These columns CogentTM TYPE-CTM silica canLC bephases usedhave for the ability to retain polar solutes at high concentrations reverse (RP) or normal of organic solvent by aqueousphase normal-phase (ONP)(ANP) modeand non-polar compounds under reversed-phase (RP) as well as ANP.conditions. These revolutionary columns use patented bonding technology to create a surface populated by siliconUse typical hydride functional groups instead of silanols. The lack method development of surface silanols leads to fast equilibration times, strategy when using lifetimes excellent peak shape and extended column these columns for a wide range of analytes. These application notes RP or ONPofmodes. demonstrate the in unique abilities Cogent TYPE-C silica LC columns for a range of clinical analysis applications. Further application notes are available at www.MTC-USA.com or from Hichrom Limited at [email protected] Cogent TYPE-C columns can be operated in 3 modes of chromatography: reversed-phase (RP), normal-phase (NP) and aqueous normal-phase. The surface silanols that are present in all Type A and B silicas, even after bonding and extensive endcapping, form a strong association with water resulting in a ‘hydration shell’ surrounding the silica. However, the silica hydride particles of TYPE-C silica are only slightly hydrophobic and therefore have a weak attraction for water allowing them to be used in aqueous normal-phase (ANP) mode, which unlike HILIC, does not require a ‘water-rich’ environment in order to operate. com retention begins depends on the solute as well as the stationary phase selected. 3. If su obta suc 90, sep Because of the attached organic group that is part of the stationary phase, the Type-C columns can also retain nonpolar compounds based on the typical reversed phase mechanism. The diagram below illustrates how this dual retention capability works for both polar (metformin) and nonpolar (glyburide) compounds: 4. In a rete colu pha com acet rete Aqueous Normal Phase (ANP) and Reversed-Phase (RP) Separations 25 Retention Time (mins) RETENTION TIME Using Aqueous Normal Phase (ANP) 20 REVERSED PHASE ANP 5. If th con tech 15 10 5 0 40 50 60 70 80 90 100 % Acetonitrile in eluent MOBILE PHASE Figure 1. Dual RP and ANP retention capability For Cogent TYPE-C silica based phases (Bidentate C18, Bidentate C8, UDC-Cholesterol, Diamond Hydride, Phenyl Hydride, UDA, Diol and Silica-C) have the ability to operate in ANP mode which enables the retention of polar solutes at high concentrations of the organic component whilst maintaining an aqueous component in the eluent. The exact point in the composition of the eluent where ANP retention begins depends on the solute as well as the stationary phase. In addition, TYPE-C columns can also retain non-polar compounds based on a typical reversed-phase mechanism. Figure 1 illustrates the dual retention capability for both polar (metformin) and non-polar (glyburide) compounds. In this case, with an eluent composition of less than 70% acetonitrile, glyburide and metformin are both retained by a reversed-phase mechanism, with the metformin eluting first. With increasing percentages of acetonitrile, the retention of metformin increases significantly due to ANP mechanisms and now elutes after glyburide. For further technical advice and additional application notes on Cogent TYPE-C Silica LC columns, contact MicroSolv Technologies, USA, www.MTC-USA.com or global distributor Hichrom Limited, UK www.hichrom.co.uk, [email protected] www.MTC-USA.com www.hichrom.co.uk P2089-10-14-003-3000