Separation of Barbiturates

Transcription

1 Bulletin # Separation of Barbiturates - Barbital - Metharbital - Butethal - exobarbital - Mephobarbital - Pentobarbital 8 7 -C8,. mm Mobile Phase: //7 A/B/C A: ACN B: TF C: mm Ammonium phosphate, p 7. Flow rate:. ml/. Temperature: ºC Injection volume: µl Detection: nm

2 Bulletin # Separation of PT-Ao Acids - Serine - Glycine - Alanine - Aoisobutyric Acid - Aobutyric Acid - Valine 7 - Norvaline 8 7 -C8,. mm Mobile Phase: /8 A/B A: ACN B:.% Trifluoroacidic Acid, p.9 Flow rate:. ml/. Temperature: ºC Injection volume: µl Detection: nm

3 Bulletin # Separation of NSAIDs Absorbance () Acetaophen - Ketoprofen - Ibuprofen - Naproxen Time (utes) -C8, mm. mm i.d. Mobile Phase: / A/B Flow rate:. ml/. A: Acetonitrile Temperature: ºC B: Water with mm Phosphoric acid at p.7 Injection volume: µl

4 Bulletin # Separation of Anticonvulsants - Primidone - Metharbital - Mephenytoin - Phenobarbital - Phenytoin 8 8 -C8,. mm Mobile Phase: /7 A/B A: TF B: mm Ammonium phosphate, p 7. Flow rate:. ml/. Temperature: ºC Injection volume:. µl Detection: nm

5 Bulletin # Separation of pioids - Naloxone - Codeine - Ethylmorphine - xycodone 8 -C8,. mm Mobile Phase:./7. A/B A: TF B: mm Ammonium phosphate, p. Flow rate:. ml/. Temperature: ºC Injection volume: µl Detection: nm

6 Bulletin # Selectivity Comparison: PBD, DB-C8 & CARB - Ethylbenzene - p-xylene 8 PBD, DB-C8 8 CARB PBD, -C8, CARB,. mm Mobile Phase: (PBD) /, (DB-C8) 7./7./, Flow rate: (CARB)./7. A/B/C Temperature: A: ACN B: Water C: TF Injection: µl Detection: nm. ml/. ºC (PBD) ºC (DB-C8) ºC (CARB)

7 Bulletin # Parabens and Phenols Separation at ºC on Diamondbond TM -C8 Absorbance () - Uracil - Phenol - Methylparaben - Ethylparaben - Propylparaben - Butylparaben Time (utes) -C8, mm. mm i.d. Mobile Phase: /8 A/B A: ACN Flow rate: Temperature:. ml/. ºC B: mm phosphoric acid, p. Injection volume: µl Detection: nm

8 Bulletin # Separation of Beta-Blockers Absorbance () Atenolol - Metoprolol - xprenolol - Alprenolol - Time (utes) -C8,. mm Mobile Phase: // A/B/C A: ACN B: TF C: mm Ammonium phosphate, p. Flow rate:. ml/. Temperature: 7 ºC Injection volume: µl Detection: nm

9 Bulletin #8 Improved Steroid ydrocarbon Selectivity on DiamondBond TM -C8 Note: Prednisolone differs from ydrocortisone only by one hydrocarbon double bond. ma U - Prednisolone 8 a =. Rs =.9 SUPELCSIL ABZ+Plus-C8 x. mm i.d. 8 - ydrocortisone 8 a =. Rs =. DiamondBond-C8 x. mm i.d. 8 Mobile Phase: //8 A/B/C A: ACN B: TF C: mm Potassium phosphate, p 7. Flow rate:. ml/. Temperature: ºC Injection volume: µl Detection: nm

10 Bulletin # Separation of Progestogen Steroids Absorbance () Chromatogram used with permission; compounds not disclosed by customer Time (utes) -C8,. mm Mobile Phase: 7./7./ A/B/C A: ACN B: TF C: Water Flow rate:. ml/. Temperature: 7 ºC Injection volume: µl Detection: nm

11 Bulletin # Ultra Fast igh Temperature PLC on Diamondbond -C8 Norm.. 7. C T emperature: o C Flow Rate:. ml/m in. Pressure Drop: 99 bar Resolution (,):. Analysis Time:.. Norm.. 7. C T emperature: o C Flow Rate:. m l/. Pressure Drop: 9 bar Resolution (,):. Analysis Time: 7.. Norm. 8-8 C T emperature: 8 o C Flow Rate:. ml/. Pressure Drop: 9 bar Resolution (,):. Analysis T ime:. Diamondbond -C8 mm x.mm Solutes: -Barbital, -Butabarbital, -Pentobarbital, -Carbromal, -Secobarbital, -Methohexital Mobile Phase: C - Isocratic 8./8. A/B C - Isocratic./8. A/B 8 C - Isocratic 8.8/9. A/B A: Acetonitrile B: mm Ammonium phosphate, p 7. Temperature: See individual runs Injection volume:. µl Detection: nm

12 Robust Methods with igh Efficiency, Bonded-Carbon PLC Columns Technical Bulletin #8 Elena Khmelnitskaia Cabot Corporation Dwight Stoll, Peter W. Carr ZirChrom Separations Inc Loss of stationary phase, retention drift, and short column life are common problems using bonded-phase silicas, especially with aggressive mobile phases. A new bonding technology overcomes these problems by attaching C8 groups to a carbon surface with ultra-stable carbon-carbon bonds. This note shows that robust methods with high efficiency are now possible using ultra-stable bonded-carbon columns. Introduction The long-term reliability of an PLC method depends greatly on the ruggedness of the stationary phase. In bonded silicas, an Si--Si bond is used to attach functional groups to the silica surface. It is well-known that this bond is subject to chemical attack, especially at low p. The silica itself dissolves readily in aqueous mobile phases at high p. Even sophisticated silica bonding technologies have not solved this problem. The basic instability of bonded silicas causes retention drift, short column life, and frequent replacement of the column and re-qualification of the PLC system. This is expensive both in terms of actual expenditures and in terms of lost productivity. Table Reproducibility of Barbiturate Method Analyte Avg. k' St. Dev. Relative St. Dev. Barbital...% Metharbital...9% Butethal...9% exobarbital.8..% Mephobarbital.7.9.7% Pentobarbital.9..% Figure shows chromatograms for the first, th, th, and final injection. Note that this improvement in ligand stability also helps with LC/MS separations, since there is no ligand bleed to create noise in the MS baseline. ZirChrom s technical support group has extensive experience in this area, and would be happy to help you with your particular application. Bonded-Carbons Zichrom Separations, Inc. and Cabot Corporation have developed new materials using unique technology to bond functional groups directly to the surface of carbon. The surface bond is C-C, which is extremely resistant to chemical and thermal attack. The authors have run mobile phases at very high p (M Na), very low p (.M N ), and at elevated temperature (up to o C), and have not observed loss of bonded ligands. Experimental A method reliability test was set-up using 8 injections of a barbiturate mixture. A single DiamondBond -C8 column (. mm x mm) was used for all of the injections. A new mixture of analytes was prepared after each injections (analytes were purchased separately from Alltech). New mobile phase (//7 TF/ACN/ mm Ammonium Phosphate, p 7.) was also prepared fresh after each injections. Results The results show that the separation has excellent long-term stability. Table shows the average retention times for the analytes and the standard deviations. The relative standard deviations are generally % or lower. Figure : Barbiturate Method Reliability =Barbital, =Metharbital, =Butethal, =exobarbital, =Mephobarbital, =Pentobarbital References () J. J. Kirkland et. al., Anal. Chem., - (989). ZirChrom Separations, Inc. 7 Pierce Street, Anoka, MN -8-STABLE- support@zirchrom.com Visit for more application notes using ultrastable, high efficiency DiamondBond columns from ZirChrom.

13 Fast Separation of Nitrosaes Dwight Stoll and Dr. Clayton V. McNeff ZirChrom Separations, Inc. Technical Bulletin #79 Interest in the analysis of nitrosae compounds is increasing as researchers become more aware of their toxicity and presence in the environment, foods, and pharmaceuticals. The extraordinary chemical and thermal stability of zirconia-based stationary phases allows for the rapid separation of these polar, basic compounds with good peak shape and efficiency. This note shows the separation of nine low molecular weight nitrosaes using a DiamondBond -C8 column. Introduction N-nitrosaes are present in both food and the environment, and have been shown to be highly carcinogenic, with toxic levels as low as micrograms per kilogram. The nitrites and nitrates commonly used as preservatives in food can cause the reaction of secondary aes to form N-nitrosaes (). Detection of these N-nitrosaes has been demonstrated using both UV detection at nm () and mass spectrometry () coupled to liquid chromatography, however these separations are typically quite lengthy, ranging from - utes. The excellent chromatographic selectivity and thermal stability of zirconia-based phases allows much faster separation of these compounds at either high or low p, where the electrospray ionization of the positively charged aes for detection by mass spectrometry is facilitated. Experimental A standard mixture of N-nitrosaes obtained from Supelco contained the following nine compounds: dimethylnitrosae, ethylmethylnitrosae, diethylnitrosae, dipropylnitrosae, dibutylnitrosae, diphenylnitrosae, nitrosomorpholine, nitrosopiperidine, nitrosopyrrolidine. The mixture was separated at 7 o C using a DiamondBond-C8 column using the following chromatographic conditions: Mobile Phase: Flow rate: Injection Vol.:. µl Detection: UV at nm. mm x mm DiamondBond -C8.-9% B from - utes A = mm Ammonium hydroxide, p 9. B = ACN. ml/. Even at a modest temperature of 7 o C the separation is rather fast, with full resolution of all nine compounds in just over utes. VWD A, Wavelength= nm (METDEVEL\.D)... Figure : Separation of nitrosaes at 7 o C. We note that our analysis shows ten peaks for the nine compound mixture. We believe peak number four in this separation may be an impurity in the mixture. Note that even temperature-sensitive compounds can benefit from modest increases in temperature, making faster analysis possible. ZirChrom s technical support group has extensive experience in this area, and would be happy to help you with your particular application. ZirChrom columns combine high efficiency with improved stability for extraordinary separations. The authors thank Dr. Mourad Rahi of Pace Analytical for the Nitrosae sample and helpful discussion. References () L. Cardenes et al. J Chrom.,. A, ; Vol. 9, pp -. () G. Bellec et al. J. Chrom,. A, 99; Vol. 77, pp 8-9. () D. Volmer et al. Rapid Comm. In Mass Spec., 99; Vol,, pp ZirChrom Separations, Inc. 7 Pierce Street, Anoka, MN -8-STABLE- support@zirchrom.com 8 9 mi Visit for more application notes using ultra-stable, high efficiency ZirChrom columns.

14 Fast Separation of Androsterone Steroids on -C8 Clayton McNeff, Ph.D. and Dwight Stoll ZirChrom Separations, Inc. Technical Bulletin # 8 This application note shows the separation of four closely related anabolic steroids (androsterone, epiandrosterone, etiocholanolone and epietiocholanolone) using a DiamondBond -C8 column. A typical analysis of these compounds involves derivatization and subsequent quantitation by GC-FID or GC-MS, however these methods tend to be labor intensive, and analytically unreliable (). Baseline resolution of all four compounds was obtained on DiamondBond -C8 at slightly elevated column temperature in under utes using isocratic elution. C C We report here a method that capitalizes on the unique temperature stability and surface chemistry of a zirconia-based stationary phase to achieve baseline resolution of these compounds in less than utes. Absorbance () 8 C C Epietiocholanolone Etiocholanolone Time (Minutes) C C C C Androsterone Epiandrosterone Figure : Structures of androsterone steroids. Figure : Separation of =Epietiocholanolone, =Etiocholanolone, =Androsterone, =Epiandrosterone on Diamondbond -C8 at ºC with the Metalox -C column heater. This method can be tailored to your specific application needs. ZirChrom method developers can help to optimize and transfer this method to your site. Please contact ZirChrom technical support at -8-STABLE- or support@zirchrom.com for details. ZirChrom phases offer unique selectivity, high efficiency, and excellent chemical and thermal stability. Introduction The rapid and accurate detection of anabolic steroids is crucial in today s sporting world. istorically the structural similarity of these compounds has made quantitative analysis by reversed-phase PLC difficult at best. These steroids are very difficult to separate on silica DS phases due to their size and structure similarities and their nearly identical mass spectra. Experimental A mixture of androsterone steroids (see Figure ) was separated at ºC using a Diamondbond -C8 column and a Metalox -C column heater. The separation conditions were as follows: Diamondbond -C8, mm x. mm i.d. (Part Number: DB-) Mobile Phase: / acetonitrile/water Temperature: ºC with Metalox -C column heater Flow Rate: ml/. Injection Vol.: µl Pressure Drop: 8 bar Detection: UV at nm References () A. Leinonen et al., J. Mass Spectrometry; 7, 9-98 (). ZirChrom Separations, Inc. 7 Pierce Street, Anoka, MN -8-STABLE- support@zirchrom.com Visit for more application notes using ultrastable, high efficiency ZirChrom columns.

15 LC/MS/MS Method for Quantifying N-Nitrosaes on -C8 Clayton McNeff, Ph.D. and Steven Rupp ZirChrom Separations, Inc. Technical Bulletin # 9 This application note shows the separation of nine pharmaceutical packaging related N-nitrosaes using a DiamondBond -C8 column. Detection is by a mass spectrometer using Multiple Reaction Monitoring (MRM) mode of the characteristic MRM transition of each individual compound. The nine N-nitrosaes are quantitated by comparison to a standard curve. The method is suitable for the analysis of N-nitrosae extractable/leachable at sub-ppb level for pharmaceutical containers. Temperature: Flow Rate: Injection: Detection: ºC with Metalox -C column heater. ml/.. ng LC/MS/MS These chromatographic conditions capitalize on the unique temperature stability and surface chemistry of zirconia-based stationary phases to achieve baseline resolution of these compounds in less than utes. Introduction PPD Development, Inc. ( provides complete bioanalytical and GMP services for drug development. Bioanalytical laboratories are located in Madison, WI and Richmond, VA. A GMP laboratory is located in Madison, WI. PPD method development experts came to ZirChrom looking for assistance in developing an approach to quantify nine structurally similar N-nitrosaes in pharmaceutical packaging. Collaborative efforts led to the development and validation of the following LC/MS/MS method. 8 NDMA Table : Method Detection/Quantification Limits. ng/ml (ppb) in water Compound LD LQ N-Nitrosodimethylae (NDMA).. N-Nitrosodiethylae (NDEA).. N-Nitrosomethylethylae (NMEA).. N-Nitrosodi-n-propylae (NDPA).. N-Nitrosodi-n-butylae (NDBA).. N-Nitrosodiphenylae (NDFA).. N-Nitrosomorpholine (NMR).. N-Nitrosopiperidine (NPIP).. N-Nitrosopyrrolidine (NPYR).. NMEA NPYR NDEA NPIP NMR NDPA NDBA NDFA Time, Figure : MRM chromatograms of nine N-nitrosaes at. ng injection. This method can be tailored to your specific application needs. ZirChrom method developers can help to optimize and transfer this method to your site. Please contact ZirChrom technical support at -8-STABLE- or support@zirchrom.com for details. ZirChrom phases offer unique selectivity, high efficiency, and excellent chemical and thermal stability. Experimental A mixture of N-nitrosaes (see Table ) was separated at ºC using a DiamondBond -C8 column and a Metalox -C column heater. The separation conditions were as follows: DiamondBond -C8, mm x. mm i.d., micron (Part Number: DB--) Mobile Phase: Gradient elution Time % A % B A:.% (v/v) formic acid B: acetonitrile Acknowledgements T. J. Deng, Prasanna Sunthankar and Aryo Nikopour, PPD Development, Inc., (Madison, Wisconsin, USA) ZirChrom Separations, Inc. 7 Pierce Street, Anoka, MN -8-STABLE- support@zirchrom.com Visit for more application notes using ultrastable, high efficiency ZirChrom columns.