Applications Notes - Direct Plasma Analysis of Drug Compounds Using Onyx Monolithic Columns

USA(310) 212-0555(310) 328-7768info@phenomenex.comtel.:fax:email:Puerto Rico(800) 541-HPLC(310) 328-7768info@phenomenex.comCanada(800) 543-3681(310) 328-7768info@phenomenex.comFrance01 30 09 21 1001 30 09 21 11franceinfo@phenomenex.comUnited Kingdom01625-50136701625-501796ukinfo@phenomenex.comIreland01 247 5405+44 1625-501796eireinfo@phenomenex.comGermany06021-58830-006021-58830-11anfrage@phenomenex.comNew Zealand09-478095109-4780952info@phenomenex.co.nzAustralia02-9428-644402-9428-6445info@phenomenex.com.au HPLC TN-1033Direct Plasma Analysis of Drug Compounds Using Onyx Monolithic ColumnsScott Waite, Michael McGinleyPhenomenex, Inc., Torrance, CA, USAIntroductionThe tandem use of liquid chromatography and mass spectrometry has become the principal mode of pharmaceutical analysis, especially for high throughput analysis employed in DMPK studies. Rapid analytical data generation is pivotal during these early stages of drug discovery. Analytical chemists have to deal with matrices such as plasma, serum, urine, or even whole blood, requiring time consuming sample preparation/clean-up prior to LC/MS analysis meant to eliminate matrix components which cause rapid decline in HPLC column performance and have deleterious effects on quantitative analysis. Solid phase extraction (SPE) and liquid extraction (LLE) are the preferred sample pretreatment techniques since they are amenable to automation, more efficient, and less labor intensive compared to other sample preparation methods. However, the constant push for faster analysis times has stimulated investigation in alternative, more rapid approaches to sample preparation. A viable and increasingly popular alternative to off-line SPE is to perform the sample clean up on-line, coupled to an LC/MS that facilitates complete automation of the whole operation of pretreatment and analysis. Such on-line extraction methods allow for the direct injection of biological samples for LC/MS analysis, improves automation, and maintains HPLC column performance. In this study, we present an evaluation of using Onyx Monolithic columns for direct injections of serum samples spiked with a mix of 11 different pharmaceuticals. We demonstrate consistent and reproducible performance with over one hundred direct serum injections. Materials and MethodsAnalyses were performed using an Agilent 1100 HPLC system, Bruker Esquire 2000 Ion-Trap MS analyzer, and a Rheodyne LabPro 10 port, 2 position-switching valve. The HPLC column used was an Onyx Monolith C18, 50 x 4.6 mm. All standards used were purchased from Sigma Chemicals (St. Louis, Missouri). Solvents were purchased from Fisher Scientific (Fairlawn, New Jersey). Column temperature was maintained at 30 °C.Table 1.*Valve position 2, HPLC flow is diverted to waste; valve position 1, HPLC flow is diverted to mass spectrometer.Time Mobile Phase AMobile Phase BFlow Rate (mL/min)Valve Position*0.00 95 5 4.0 20.75 95 5 4.0 20.76 95 5 1.0 15.00 15 85 1.0 15.01 95 5 4.0 2HPLC GradientMobile Phase A: 0.1 % Formic acid in WaterMobile Phase B: 0.1 % Formic acid in AcetonitrileSerum samples were diluted 2:1 with 0.1 % formic acid and spiked with 11 pharmaceutical drug compounds at a concentration of 200 ng/mL. Aliquots of 10 µL of diluted serum samples were directly injected into the Onyx Monolithic C18 for MS analysis. The flow rate for loading sample was set at 4 mL/min for 0.75 minutes with the switching valve diverting flow to waste. The valve is then switched to position 1 for the mass spectrometer, flow adjusted to 1 mL/min and a gradient of 5 % - 85 % mobile phase B initiated to elute and separate the analytes. The valve is switched back to position 2 for equilibration of initial starting conditions. MS Conditions:• ESI in positive ion mode • Nebulizer set at 60 psi• Dry Gas set at 12 L/min • Dry Temp set at 365 °CResults and DiscussionThis work examined the use of Onyx Monolithic columns for the direct injection of diluted serum samples for LC/MS analysis. The dual role of the monolithic column is both to remove the matrix macromolecules (as a trapping column) and to maintain chromatographic performance for the elution and separation of analytes (as an analytical column). Compared to a traditional particle packed silica column, the monolithic column generates a much flatter Van Deemter plots at high flow rates due to the better mass-transfer properties allowing for faster HPLC separations without a noticeable effect on chromatographic resolution. However, running high flow rates directly into the MS source will result in lower signals. To compensate for this, either a post-column split can be used or the flow rate can be adjusted during the elution step for optimal conditions for detection as is summarized in Table 1. Reproducibility and ruggedness was demonstrated by sequential injections of 100 serum samples with apparent recoveries greater than 90 %. Figure 1 shows overlay of five sequential injections of drug mix in serum. Figure 2 shows individual chromatograms of test compounds in serum samples.Figure 1. Overlay of 5 sequential injections demonstrating reproducibility.Figure 1: Overlay of 5 sample injections. Pharmaceutical compounds are spiked into a plasma sample and selected ions are monitored by MS. Results indicate that repetitive dilute-and-shoot injections show little effect on the chromatography and recovery of key analytes.?????? ??? ??? ??? ??? ??? NJO?????????*OUFOT?Y???