Using High Temperature HPLC for Improved Analysis in the Pharmaceutical Industry

Transcription

1 Using High Temperature HPLC for Improved Analysis in the Pharmaceutical Industry Stephanie J. Marin Brian Jones, Dale Felix, Jody Clark Selerity Technologies, Inc. Salt Lake City, UT

2 Abstract It is now becoming recognized that operating HPLC systems at higher than ambient temperatures results in improved peak shape and faster runs without sacrificing efficiency. Because viscosity is significantly reduced at higher temperatures, columns packed with smaller diameter particles can now be used without a significant pressure penalty. Consequently, combining the small diameter packings with short column lengths result in highly efficient, extremely fast separations. High Temperature Liquid Chromatography (HTLC) thus meets the demanding high throughput performance requirements of the pharmaceutical industry.

3 Introduction High Temperature Liquid Chromatography (HTLC) offers several advantages to the separation scientist. Temperature programming can replace solvent gradients with thermal gradients. Faster and more efficient separations are achieved. Adequate preheating of the mobile phase is critical to avoid thermal mismatch band broadening. This poster demonstrates the use of HTLC to analyze a number of pharmaceutical samples including an active pharmaceutical ingredient. Samples that normally require a binary solvent gradient were separated using a programmed temperature gradient and an isocratic mobile phase.

4 Better Chromatography with Temperature Gradient Programming Change retention through temperature gradient programming Replace solvent gradients with temperature gradients Water less polar and behaves more like methanol so less organic modifier needed Faster and More Efficient Separations Higher efficiency - better resolution Increased diffusion rates provide a reduction in plate height at higher temperature Lower viscosity and back pressure permits higher flow rates with smaller particle size packings Speed Flatter van Deemter curves allow operation at flow rates many times optimal velocity

5 Temperature Effects on Plate Height h (x 10-4 ) ºC 80 ºC 120 ºC 150 ºC u (cm/s) Effect of temperature on plate height. The Van Deemter curve flattens out as the temperature is increased. This means that operation at higher linear velocities at elevated temperature does not sacrifice efficiency as significantly as it does at ambient temperature.

6 Selerity Polaratherm Series 9000 Total Temperature Controller Forced air oven and chiller Isothermal and thermal gradient operation Sub-zero to 200 C Flow rates up to 10.0 ml/min Thermal gradients up to 30 C/min Mobile phase preheating and pre-cooling Peltier effluent temperature control Vapor sensor Compatible with any HPLC system

7 Why is Mobile Phase Preheating so Important? No Preheating Parabolic flow caused by mobile phase heating up faster along column wall Flow With Preheating Mobile phase at column temperature eliminates parabolic flow Flow

8 Mobile Phase Preheater Very responsive and non-invasive Low-mass and low-volume: <2 grams mass (including the tubing),<1 µl totally swept volume 0.005, and ID available Can respond to fast temperature ramps used in thermal gradients

9 Mobile Phase Preheating Improves Chromatography Separation of Barbiturates 40 Zirchrom PBD, 80 C 30 Barbital Preheater Off - Thermal Mismatch mv Butabarbital Carbromal Secobarbital Preheater On Sharp Peaks Minutes

10 Examples The following examples show several pharmaceutical samples separated using thermal gradients. The first chromatogram shows the separation of steroids using water as a mobile phase. The next two chromatograms show the separation of over-the-counter analgesics using a Selerity Blaze C 8 and a Thermo HyperCarb Column. Note the complimentary selectivities of the two columns. The next chromatogram shows the separation of narcotics on the HyperCarb column. The final example is the extraction and analysis of an active pharmaceutical ingredient in a name brand drug and a knock-off formulation available on the internet.

11 Separation of Steroids Using Water as a Mobile Phase Column: Zirchrom PBD, 3 µm 100 X 4.6 mm Detection: UV 254 nm mv seconds! Flow Rate: 6.0 ml/min Mobile Phase: Water! Temperature: 200 C Flow Rate: 3.0 ml/min Mobile Phase: 25:75 acetonitrile:water Temperature: 50 C Elution Order: Uracil Androstadienedione Androstenedione Epitestosterone Minutes

12 Separation of Analgesics on a Selerity Blaze C 8 Using a Thermal Gradient mv Column: Selerity Blaze C 8, 3 µm 100 x 4.6 mm Mobile Phase: 40:60 acetonitrile:water with 0.1%TFA Flow Rate: 1.5 ml/min Detection: UV 220 nm Temperature Program: hold at 50 C for one minute, ramp to 100 C at 30 C/min, hold six min Minutes Elution Order: Acetaminophen Caffeine Salicylamide Aspirin Salicylic acid Ibuprofen Naproxen

13 Analgesics Using a Hypercarb Column mv and a Thermal Gradient Minutes Column: Thermo Hypersil-Keystone Hypercarb, 7 µm, 100 x 4.6 mm Mobile Phase: 35:65 acetonitrile:water with 0.1% TFA Flow Rate: 4.0 ml/min Detection: UV 220 nm Temperature Program: thermal gradient from 125 to 200 C at 30 /min, hold five min. Elution Order: Caffeine Aspirin Salicylic Acid Ibuprofen Phenacetin Acetaminophen Naproxen

14 Separation of Narcotics on Hypercarb Column Using a Thermal Gradient mv Minutes Column: Thermo Hypersil-Keystone Hypercarb, 7 µm, 100 x 4.6 mm Mobile Phase: 50:50 acetonitrile:50 mm ammonium acetate ph 9.0 Flow Rate: 1.0 ml/min Detection: UV 220 nm Temperature Program: hold at 50 C for two minutes, ramp to 150 C at 30 /min, hold ten min. Elution Order: Morphine Hydromorphone Codeine Methadone Hydrocodone Oxycodone Diacetylmorphine

15 Extraction and Analysis of Sildenafil Citrate in Viagra and an Offshore Formulation This example shows the extraction and HTLC analysis of Sildenafil Citrate in Pfizer s Viagra and in a Sildenafil tablet available on the internet from a company based in Belize. The Belize tablets claim to be comparable to Viagra at a much lower cost. We extracted the tablets using a procedure recently published in the literature and analyzed the extracts using a thermal gradient and UV detection.

16 Sample Prep for Sildenafil Analysis In 100 mg Viagra and Offshore Tablet Ground tablet in mortar and pestle Extracted with 30 ml 50:50 methanol:water by sonicating for 20 minutes Centrifuged at 3000 rpm for 15 minutes Diluted supernatant to 100 ml with 50:50 methanol:water (rinsed three times) Filtered through 0.45µm nylon prior to injection Tseng and Lin, J. of Food and Drug Analysis, 10, (2002)

17 Pfizer Viagra Offshore Tablet

18 Analysis of Sildenafil in Viagra and Offshore Tablet Using Selerity Blaze C 8 mv Column: Selerity Blaze C x 4.6 mm, 3µm Mobile Phase: 35:65 acetonitrile:water with 0.1% TFA. Flow Rate: 1.0 ml/min Detection: UV 220 nm Temperature Program: hold at 40 C for two minutes, ramp to 100 C at 15 /min, hold for six min Sildenafil Citrate in Viagra Sildenafil Citrate in Offshore Tablet Minutes

19 How much Sildenafil Citrate Does the Offshore Formulation Contain? Assume Pfizer Viagra tablet contains 100 mg of Sildenafil Citrate Compare peak areas Correct for amount of tablet used Offshore tablet contains about 80 mg of Sildenafil Citrate Notice that there appears to be no evidence of analyte degradation due to exposure to high temperature.

20 Conclusions Thermal gradients can replace solvent gradients for analysis of many pharmaceuticals Increasing the temperature reduces the amount of organic modifier needed Shorter analysis times with better efficiency result from higher temperatures Use authentic Pfizer Viagra

21 Acknowledgements The authors thank Thermo Hypersil-Keystone and Hamilton Company for providing columns for this work Turn up the Heat!