Method Development of LC-MS/MS Analysis of Aminoglycoside Drugs: Challenges and Solutions authors Angela (Qi) Shen, Ling Morgan, Marcele L. Barroso, and Xin Zhang; Tandem Labs Tuyen Nguyen; Sepracor Inc. introduction The aminoglycosides are mainstream drugs used in the treatment of serious gram-negative systemic infections. They irreversibly bind to the ribosome site and other cellular structures in order to interrupt the protein synthesis. The common feature of these drugs is an aminocyditol ring linked to an amino sugar in the structure. Therefore, these drugs are highly soluble in water, exhibit low plasma protein binding, and are more than 90% excreted through the kidney. Herein, we share our experiences with LC-MS/MS method development for the analysis of aminoglycosides in plasma samples with HILIC and ion pairing chromatography. OBJECTIVE To develop a reliable and generic LC-MS/MS method to quantify the aminoglycoside class of drugs. approaches Sample Generation 1. Sample Matrix: Mouse, Rat and Guinea Pig Plasma with K 2 EDTA as the anti-coagulant. (Spiking solution approach was employed in the preparation of all standard and QC calibrants.) 2. Sample Treatment: Acid treated plasma samples underwent protein precipitation with a ratio of 1:6, Sample:Acetonitrile. (Cold internal standard prepared in Acetonitrile was preferred to generate cleaner sample extract for LC-MS/MS analysis.) The transferred supernatant was dried down and reconstituted with 1% Formic Acid (FA) in water, or initial MPA/MPB composition of LC conditions. 3. Internal Standard: Vancomycin and glyburide. Angela (Qi) Shen et al. (2008) 1
LC Conditions Two types of LC conditions were employed for the analysis: ion pairing chromatography and HILIC. (See details in each respective section.) MS Conditions MRM mode and electrospray ionization mode (ESI), in positive, were employed for all analytes. Amikacin: 586.7 > 163.2; Streptomycin: 582.7 > 263.2 Spectinomycin: 333.2 > 98.0; Gentamicin: 478.6 > 160.3 Vancomycin: 724.8 > 144.2; Glyburide: 494.5 > 369.1 Data Acquisition API4000 or API5000, utilizing ESI mode, were needed for required LLOQs for each analyte under different experimental conditions. Analyst 1.4.2 was used for regression and linearity calculations.results AND DISCUSSION Figure 1. Model Compound Chemical Structures Angela (Qi) Shen et al. (2008) 2
results and discussion Sample Preparation Nonspecific Binding: Initially, an inconsistent LLOQ was obtained during amikacin analysis. Investigational experiments were conducted and revealed non-specific binding for all four compounds. Table 1. A brief summary of the situations tested 1. 2. 3. All four compounds exhibit severe binding to glass vials after four consecutive transfer steps among four regular 4mL glass vials. All four compounds bind to plastic tubes. Gentamicin and amikacin are more severe than the other two. Addition of organic acid, such as 1% FA in stock solution eases the non specific binding to glass vials, as well as plastic tubes and 96 well plates. Acid Treatment Simple protein precipitation was used with a ratio of 1:6, Sample:Acetonitrile. (Cold internal standard in Acetonitrile solution is preferred to generate cleaner sample extract for LC-MS/MS analysis.) Acidification of samples prior to PPT proved to be the key step for good recovery. Angela (Qi) Shen et al. (2008) 3
Figure 2. Initial Result of Treatment of Plasma Sample with Acid Optimization: After a series of experiments, 10% v:v of 3N HCl was determined to be a suitable choice for minimum sample volume of 25µL. After PPT, the sample supernatant was transferred and dried down. Initial LC composition of MPA/MPB was determined the preferred choice for the re-con solution Figure 3. Selective Ion Pairing Chromatograms: untreated versus acid treated Angela (Qi) Shen et al. (2008) 4
HILIC Condition: Mobile Phases: 1. MPA: 5 or 25 mm Ammonium Formate in Milli-Q water with Formic Acid (ph = ~2.5) 2. MPB: Acetonitrile with 1% (v:v) Formic Acid HPLC Column: SeQuant Zic-HILIC 50 X 2.0 mm, 5 µm particle size. Flow Rate: 600 µl/min Gradient Profile: Hold initial 90% of MPB for 0.5 minutes; decrease to 10% of MPB at 1.0 minute; retain at 10% of MPB for 3 minutes; return to 90% of MPB for column conditioning. Figure 4. HILIC: Impact of Different Concentrations of Ammonium Formate: 20 mm vs 5 mm (ion suppression) Decreasing the concentration of Ammonium Formate from 20 mm to 5 mm in MPA improved the MS response for all analytes due to ion suppression reduction. On the other hand, such change had negative impact on the chromatography for gentamycin analysis. Angela (Qi) Shen et al. (2008) 5
Table 2. Summary of LLOQ for Four Compounds Under Different Conditions Ion Pairing Chromatography 1. 2. Mobile Phases: MPA: 10 mm Nonafluoropentanoic Acid with 10 mm Ammonium Hydroxide in Milli-Q water. MPB: 5 mm Nonafluoropentanoic Acid with 5 mm Ammonium Hydroxide in 10:90 mixture of Milli-Q water:acetonitrile HPLC Column: Agilent Zorbax SB-C8 30 X 2.0 mm, 5 µm particle size. Flow Rate: 500 µl/min Gradient Profile: Hold initial 20% of MPB for 0.5 minutes; increase to 90% of MPB for 2.4 minutes; retain 90% of MPB for 0.5 minutes; return to 20% of MPB for column conditioning. Figure 5. Ion Pairing Chromatography Angela (Qi) Shen et al. (2008) 6
The major advantage of the employment of ion pairing chromatography is the improvement of the detection limit for all analytes by 10 fold (see Table 2). In addition, ion suppression phenomenon is manageable (data is not shown here). We did observe column related carry-over issues. The detail is summarized in Table 3 below. Table 3. Summary of Carryover Evaluation conclusion We report two different LC-MS/MS approaches for the analysis of aminoglycosides using ion pairing and HILIC. Care must be taken during the sample preparation step, since severe non-specific binding to glass and some binding to plastic were observed. Pre-treating the sample with acid is critical for good compound recovery from the samples. Both ion pairing and HILIC demonstrate good chromatography for majority of compounds tested. We also observed that ion pairing chromatography demonstrated better detection limits for three of the compounds. Both ion pairing and HILIC have comparable detection limits for Spectinomycin. Further optimization of HILIC conditions to improve sensitivity is needed for all compounds. Angela (Qi) Shen et al. (2008) 7
Tandem labs Tandem Labs is a leading bioanalytical and immunoanalytical contract research organization (CRO) supporting pharmaceutical and biotechnology companies with their discovery, preclinical, and clinical drug development programs. Our laboratory menu includes advanced mass spectrometry, immunoanalytical support, pharmacokinetics, and pharmacodynamics. We also provide clinical research services through our strategic partner. Download more posters and white papers at our website www.tandemlabs.com/capabilities_publications Find full contact information online www.tandemlabs.com/contact Tandem Labs-Utah 1121 East 3900 South Building C, Suite 105 Salt Lake City, UT 84124 (801) 293-2400 Tandem Labs-New Jersey 115 Silvia Street West Trenton, NJ 08628 (609) 434-0044 Tandem Labs-New England 35R Cabot Road Woburn, MA 01801 (781) 933-2769 x 123 Angela (Qi) Shen et al. (2008) 8