Improvements in Sensitivity for Quantification of Steroid Phosphate Drugs Using ACQUITY Premier System and ACQUITY Premier Columns

Analytes with electron rich moieties such as phosphate and carboxylate groups are susceptible to chelation with metal surfaces across the chromatographic system and column. This often results in poor peak shape, reduced sensitivity, with poor robustness and reproducibility, all of which can adversely affect the development of a successful bioanalytical method. The ACQUITY Premier System and ACQUITY Premier Columns with MaxPeak High Performance Surface (HPS) mitigate this problem by providing a chemically inert hybrid organic-inorganic surfaces which prevents metal chelation without affecting chromatographic selectivity.

In this application note, we show the impact of using this novel surface for quantifying hydrocortisone phosphate and dexamethasone phosphate in extracts of human plasma. Using the ACQUITY Premier System and ACQUITY Premier Columns, we were able to achieve a 10-fold improvement in the lowest limit of quantification (LLOQ) for hydrocortisone phosphate and 7.5-fold increase in LLOQ for dexamethasone phosphate. Additionally, better overall chromatographic performance resulted in better peak shapes and robust and reproducible integration of peaks, especially at the low concentrations.

Benefits

Superior peak shape, simpler peak integration, and improved sensitivity

Bioanalysis laboratories constantly strive for more sensitivity as they try to better define the pharmacokinetics (PK) of candidate drugs at low systemic concentrations or are required to use lower sample volumes to perform analysis in pre-clinical or pediatric studies. Over the years, continuous advancements in MS instrumentation and sample extraction procedures have allowed scientists to achieve lower limits of quantification enabling them to more deeply investigate their drug of interest. However, current instrumentation platforms still pose major challenges for certain analyte classes where metal chelation can be problematic. Examples of this are certain compounds containing phosphate groups (e.g., ATP), uncharged amines, and deprotonated carboxylate groups (citrate, lactate, etc.,) that are electron rich and are easily adsorbed onto metal surfaces such as the stainless steel used in LC systems. This can have a deleterious effect on bioanalytical methods resulting in complicated method development, time consuming data analysis, insufficient assay sensitivity, and poor reproducibility. In attempting to address these issues bioanalyst’s have employed chelating reagents as mobile phase additives, PEEK connection tubing and system passivation.¹ However, these solutions are not ideal as they are either non-permanent, incompatible with solvents such as DMSO or reduce ionization efficiency for the analytes causing reduced sensitivity.¹

To address these challenges, Waters has developed a class of new technologies, known as MaxPeak High Performance Surfaces (HPS). MaxPeak HPS in ACQUITY Premier are comprised of a resilient, highly crosslinked layer that is chemically similar to bridged-ethyl hybrid silica.¹ The MaxPeak HPS provides a highly effective surface barrier that mitigates against undesired interactions with metal surfaces. Here, we highlight the benefits of the ACQUITY Premier Solution with MaxPeak HPS for bioanalysis using hydrocortisone phosphate and dexamethasone sodium phosphate as exemplars. Dexamethasone phosphate and hydrocortisone phosphate are anti-inflammatory corticosteroids used in the treatment of endocrine disorders as well as immune and allergic conditions such as arthritis, psoriasis, lupus, and ulcerative colitis.² Dexamethasone phosphate was also recommended for COVID-19 patients with severe respiratory symptoms.³ Therefore, there is exceptional interest in improving the sensitivity of detection for these compounds.

Sample Preparation

Lyophilized hydrocortisone phosphate and dexamethasone phosphate were dissolved in DMSO to make stock solutions at 1 mg/mL. These stocks were further diluted in 5% methanol in water to make working standards at 100 µg/mL and 10 µg/mL. These working standards were then spiked into human plasma to generate a calibration curve from 5–1000 ng/mL for hydrocortisone phosphate and 1–1000 ng/mL for dexamethasone phosphate. 100 µL of each sample were transferred to a micro-centrifuge tube and extracted using 300 µL of methanol. The samples were vortexed and centrifuged at 13,000 rcf for 10 min. 200 µL of the supernatant was transferred to LC-MS vials and used for analysis.

Method Conditions

MRM methods for both analytes were developed using QuanOptimize. A short LC method with a generic gradient was used for analysis. The method details are listed below.

Due to the physico-chemical characteristics of hydrocortisone phosphate and dexamethasone phosphate, these analytes are susceptible to adsorption on stainless steel surfaces. This phenomenon is exacerbated at low concentrations as most or all the analyte can be lost to the needle, transfer tubing, pre-heaters, and column hardware. Mitigating these interactions through the use of chemically inert surfaces along the flow path has the potential of increasing the amount of analyte that reaches the detector, thereby improving the lower limit of quantification (LLOQ) that can be achieved, as well as increasing the precision of the assay.

To test this hypothesis, calibration curve and QC samples were extracted using the procedure described above and injected on the ACQUITY Premier System with an ACQUITY Premier Column as well as a conventional ACQUITY UPLC System with a standard ACQUITY UPLC Column. 

As seen in the top chromatogram in Figure 1A, using the ACQUITY Premier System with an ACQUITY Premier Column, we achieved a LLOQ of 5 ng/mL with signal-to-noise of >15 for hydrocortisone phosphate. The lower chromatogram of Figure 1a shows the same sample injected on a conventional ACQUITY LC system and UPLC column. The peak in the lower chromatogram has a much lower intensity with a signal-to-noise <3 and may have much higher inter- and intra-day CVs which can affect the reproducibility, especially for low concentration samples. Similarly, as shown in the top chromatogram of Figure 1B, we achieved a LLOQ of 1 ng/mL with a signal-to-noise >10 for dexamethasone phosphate. However, the chromatogram of the same sample injected on the conventional LC system shows a peak with a significantly lower intensity as shown in the lower chromatogram of Figure 1B with a signal-to-noise of <4. Figure 1C compares the response obtained on the two systems at 50 ng/mL for hydrocortisone phosphate, which is the lowest concentration that could be accurately quantified on the standard ACQUITY LC system with standard ACQUITY columns. In addition to having a significantly higher peak area response, the signal observed on the ACQUITY Premier System/Column is more symmetrical in shape and therefore more reproducibly integrated, compared to the peak observed on the standard hardware using the same integration parameters. Figure 1 compares the chromatograms for dexamethasone phosphate at 7.5 ng/mL, which is the observed lower limit on the standard system and column combination. The peak area responses and peak shapes for both systems are similar. However, concentrations below 7.5 ng/mL injected on the standard system/column either showed no peaks at all or provided irreproducible peaks which cannot be accurately integrated. This lack of reproducibility may be attributed to the analyte being adsorbed on the system. On the ACQUITY Premier System with ACQUITY Premier Columns, all concentrations from 7.5 ng/mL down to 1 ng/mL showed well defined chromatographic peaks which could be accurately integrated and showed a linear increase in response with increase in concentration.

Using the ACQUITY Premier System and ACQUITY Premier Columns, we achieved a linear calibration curve for hydrocortisone phosphate from 5–1000 ng/mL with r² > 0.993, and for dexamethasone phosphate from 1–1000 ng/mL with r² > 0.992 using a weighting factor of 1/x (Table 1A). The slope and intercept for hydrocortisone phosphate were 0.043 and 0.054, respectively; and for dexamethasone phosphate were 0.119 and 0.129, respectively. The closeness of these values to 0 implies negligible bias of the calibration lines and therefore more accurate quantification of unknown samples when using these calibration curves. This is confirmed by the accuracy and precision statistics calculated for both analytes at the LLOQ and the low, mid, and high QC levels. For hydrocortisone phosphate, accuracies of between 98–102% and precision of <11% were observed (Table 1B). For dexamethasone phosphate, accuracy and precision values were between 92–112% and <8%, respectively (Figure 1C).  

Representative chromatograms at the low, mid, and high QC levels for hydrocortisone phosphate (Figure 2A) and dexamethasone phosphate (Figure 2B) show a linear, concentration dependent, increase in area counts for both analytes.

Quantifying metal sensitive compounds on standard LC and column hardware suffers from poor peak shape and lack of reproducibility, resulting in an inability to achieve low LLOQs.

Using the ACQUITY Premier System with ACQUITY Premier Columns for hydrocortisone phosphate and dexamethasone phosphate we achieved:

• Sensitivity improvements of 10x (hydrocortisone phosphate) and 7.5x (dexamethasone phosphate)
• Narrower peak widths and more symmetrical peak shapes allowing for more reproducible integration, especially at the lower concentration levels
• Assay precision of <11% (hydrocortisone phosphate) and <8% (dexamethasone phosphate) across LLOQ, LQC, MQC, and HQC levels

1. Hassn M. E. Dexamethasone in Severe COVID-19 Infection: A Case Series. Respiratory Medicine Case Reports. 2020, 31, 101205.
2. Cevc, G.; Blume, G. Hydrocortisone and Dexamethasone in ery Deformable Drug Carriers Have Increased the Biological Potency, Prolonged Effect, and Reduced Therapeutic Dosage. Biochimica et Biophysica Acta (BBA) – Biomembranes. 2004, 663 (1–2), 61–73.
3. Lauber, M.; et al. Low Adsorption HPLC Columns Based on MaxPeak High Performance Surfaces. Waters White Paper, 720006930EN, 2020.