Quantitatitive Analysis of Phosphorothioate Oligonucleotide in Human Plasma Using LC-MS/MS with On-Line Extraction

Transcription

1 Laixin Wang, Sherry Liu, Qiuying Zhu, Scott Reuschel and Min Meng Tandem Labs Quantitatitive Analysis of Phosphorothioate Oligonucleotide in Human Plasma Using LC-MS/MS with On-Line Extraction Introduction Oligonucleotide therapeutics are gaining renewed confidence as a new class of drugs. Many bioanalytical methods for oligonucleotides have been developed to keep up with the pace of drug development needs. LC-MS/MS is one of the most popular techniques due to its unprecedented specificity; however, the sensitivity for most LC-MS/MS assays is still not as good as corresponding ligand binding assays for some large oligonucleotide therapeutics. To improve sensitivity and throughput, we have successfully developed a fully automated online SPE integrated LC-MS/MS assay. Methodology 1. Sample Preparation 100 µl of plasma sample, 50.0 µl of internal standard working solution and 150 µl of extraction buffer were aliquoted into the corresponding wells of a 96-well plate. The samples were sonicated for 10 minutes and stored at 4 C for analysis. 2. Online SPE Cartridge: Conditioning: Equilibration: Cartridge Wash: Elution: Cartridge Flush: Spark Holland HySphere HD 7μm Methanol 2% HFIP & 0.4% TEA in water 2% HFIP & 0.4% TEA in water Mobile phase from gradient pumps Water/MeOH 50/50 (v/v) tandemlabs.com Laxin Wang et al. (2013) 1

2 Methodology (continued) 3. LC-MS/MS Mass Spec: ABI Sciex API 5000 Source and ionization: ESI (negative ion mode) Column: C18, 2 x30 mm Column Temperature: 60 C Flow Rate: ml/min Mobile Phase: A: 2% HFIP & 0.4% TEA in water B: 2% HFIP & 0.4% TEA in MeOH LC Program: Gradient Source Temperature: 500 C SRM transitions: TL g95.0 TL0901(n-6) 631.7g95.0technically challenging. Results and Discussion Main Characteristics of Symbiosis Pharma Online Spe System Symbiosis Pharma Online SPE System is composed of two units including the autosampler and LC unit (Reliance) and the SPE extraction unit (ACE) (Figure 1). The system can be operated in two distinct modes: LC mode and XLC mode. In LC mode, the unit functions as a regular LC and autosampler. In XLC mode, on-line SPE extraction is conducted (Figure 2). Online SPE extraction can also be performed in two distinct modes: elute-and-shoot mode or peak-focusing mode. In the elute-and-shoot mode, the entire extract is injected directly onto the HPLC column; therefore, the elution solvent needs to be compatible with the HPLC column and its corresponding mobile phase. In peak focusing mode, if the elution solvent is incompatible with the mobile phases or if the peak shape is not optimal, a third solvent can be introduced post extraction in order to alter the solvent strength and ph. Figure 1. Symbiosis Pharma online SPE extraction unit Figure 2. Online SPE (XLC) mode tandemlabs.com Laxin Wang et al. (2013) 2

3 Advantages of the Symbiosis Pharma Online SPE Method Sample preparation consists only of diluting plasma sample with internal standard and buffer solution (if needed). Extraction is fully automated and integrated with chrom-atographic analysis, greatly improving overall precision and reducing the chances of human error. Samples are processed in a serial manner; i.e., while one sample is analyzed by LC-MS/MS, the next sample is extracted concurrently. Thus, there is virtually no sample preparation time. The entire extract is loaded onto the analytical column. Therefore, it can minimize the sample use or increase the detection sensitivity. There is no sample drying-down and reconstitution steps involved during the sample preparation process. This eliminates the analyte loss caused by non-specific binding and/or solubility issues. This is particularly helpful for the bioanalysis of large biopharmaceuticals, such as oligonucleotides and peptides. Online SPE cartridges are designed to be washed and reused multiple times to reduce the overall cost of analysis. However, no consensus has been reached on the acceptability of this practice from a regulatory or industry standpoint. Limitations of the Symbiosis Pharma Online SPE Method Because no final sample extract is retained, reinjection reproducibility needs to be demonstrated as diluted matrix stability. Compounds that are unstable in plasma would pose a challenge for diluted matrix stability and thus may not be suitable for online SPE extraction. Relative extraction recovery can only be conducted manually via peak fraction collection. Thus, the best solution is to conduct absolute recovery using a combination of the LC and XLC modes. Troubleshooting analytical issues such as contamination and carryover are very challenging to perform due to the complexity of the system. A systematic approach is required to eliminate each component one-by-one following vendor instructions. The integrated online extraction system needs to be maintained regularly. Sample Extraction and LC Method Development TL0901 is an 18-mer phosphorothioate DNA oligonucleotide (sequence is 5 -ACTGTACGATTCGACCTA) with a molecular weight of Daltons. A 12-mer analog phosphorothioate DNA oligonucleotide [TL0901(n-6)] was used as the internal standard for quantitations. A typical multiple charged Q1 scan of TL0901 was obtained under optimized negative ESI conditions (Figure 3). Similar product ion scan spectrum was obtained from each of the Q1 peaks (Figure 4). tandemlabs.com Laxin Wang et al. (2013) 3

4 A Spark Holland HySphere method development cartridge plate was used for initial SPE cartridge screening. It was found that the HySphere CN-SE 7 µm cartridge gave the best extraction recovery for these particular analyte and IS oligonucleotides. A 2x30mm C18, 3.5 µm, column was used as analytical column to minimize the assay carryover. TL0901 can be readily separated from the internal standard chromatographically under the experimental conditions. Figure 3. Representative Q1 scan mass spectrum of TL0901. Figure 4. Representative product ion scan spectrum of TL0901 tandemlabs.com Laxin Wang et al. (2013) 4

5 Preliminary Results A 0.5 ng/ml LLOQ was achieved by injecting 100 µl of diluted plasma samples (Figure 5). Carryover (~5% of previous sample) was observed (Figure 7). The carryover was contributed by both the system and LC columns for this assay. We are currently working on reducing carryover for this assay. The calibration curves fitted linear regression well in the range of ,000 ng/ml (Figure 10). Figure 5. Representative chromatogram of LLOQ (0.457 ng/ml TL0901 in human plasma) Figure 6. Representative chromatogram of ULOQ (1000 ng/ml TL0901 in human plasma) tandemlabs.com Laxin Wang et al. (2013) 5

6 Figure 7. Representative carryover blank (blank after ULOQ at 1,000 ng/ml) Figure 8. Representative blank tandemlabs.com Laxin Wang et al. (2013) 6

7 Figure 9. Representative blank with internal standard (0-ng/mL QC) Figure 10. Representative calibration curves in triplicates ( ,000 ng/ml) tandemlabs.com Laxin Wang et al. (2013) 7

8 Conclusion A highly sensitive and fully automated online SPE-LC-MS/MS method was developed to quantify an 18-mer phosphorothioate DNA oligonucleotide in human plasma. References 1. Wang L, Yuan W, Zhao Y, Chen J, vonarx G, Meng M and Bennett P, Quantitation of TL0901 oligonucleotide in human plasma using LC-MS/MS, AAPS Annual Meeting and Exposition, November 8-12, 2009, Los Angeles, CA. 2. Hemsley M, Ewles M and Goodwin L, Development of a bioanalytical method for quantification of a 15-mer oligonucleotide at sub-ng/ml concentrations using LC-MS/MS, Bioanalysis, 4(12), , tandemlabs.com Laxin Wang et al. (2013) 8