Abstract

Abstract

This technical note demonstrates a sensitive method for the quantitation of pramlintide in human serum on the SCIEX 7500+ system. A lower limit of quantitation (LLOQ) of 10 pg/mL (100 fg on column) was achieved with a fast runtime of 6 min (Figure 1).

Pramlintide was approved by the FDA in 2005 for the treatment of type 1 and type 2 diabetes.¹ The structure of pramlinitide is comprised of a 37-amino acid amylin analogue based on the rat amylin sequence. Typically, pramlinitide is applied as an adjunct to insulin therapy for diabetic patients who are using mealtime insulin.² It is commonly administered subcutaneously at a concentration of 30 µg.³ Considering that the administered concentrations are quite low, toxicokinetic and pharmacokinetic studies require sensitive methods to manage low circulating levels (pg/mL), quick absorption (<30 minutes) and rapid excretion (~1 hour) of amylin agonists to effectively evaluate safety and efficacy.

Figure 1. Representative extracted ion chromatograms (XICs) for pramlintide in extracted human serum at matrix blank, 10 pg/mL (LLOQ) and 25 pg/mL. No interferences were observed in the matrix blank.

image-top

Key-features

Key benefits for analysis of pramlintide using the SCIEX 7500+ system

• Sensitive quantitation of pramlintide: Achieve 10 pg/mL LLOQ for the quantitation of pramlintide in human serum.
• Low serum consumption: Perform sensitive quantitation with a 100 µL of human serum paired with a simplified SPE methodology
• Fast analysis: Perform sensitive LC-MRM analysis of pramlintide in human serum within a 6 min runtime
• Effortlessly meet critical quantitative performance criteria: Achieve accurate quantitative performance with %CV <11 at all concentration levels across a linear dynamic range (LDR) of 4 orders of magnitude
• Streamlined data management: SCIEX OS software, a 21 CFR Part 11-compliant platform, simplifies data acquisition and processing

Introduction

Introduction

Pramlintide, a synthetic version of amylin, has proline amino acids at positions 25, 28, and 29 of the human amylin protein.⁴ In contrast to amylin, it is readily soluble in water and does not aggregate, stick to surfaces, or form insoluble particles. In diabetic individuals, intravenous pramlintide lowered postprandial plasma glucose levels after a meal but not after an intravenous glucose infusion. These findings suggest that pramlintide's mode of action involves slowing glucose absorption by inhibiting gastric emptying.

Pramlintide is typically administered in low dosage and has a fast absorption, requiring a highly sensitive method to quantify at very low concentrations to assess its efficacy and safety in humans.

Methods

Methods

Standard preparation: 1 mg of pramlintide stock was procured from Medchem Express and dissolved in methanol.
The spiking solutions were prepared using 0.1% formic acid in 10:90 (v/v) methanol:water as diluent.

Sample preparation: Pramlintide was spiked into 100 µL of human serum at concentrations ranging from 10 to 100000 pg/mL. A 0.3 mL aliquot of chilled 1% formic acid in methanol was added to 100 µL of serum and vortexed for 5 min. The samples were centrifuged at 1204 rcf for 5 min. Strata X-CW was conditioned with methanol followed by 0.1% formic acid in water. The supernatant was loaded onto the conditioned cartridge and washed with water followed by 20% acetonitrile in water. The samples were eluted with 100 µL of elution solvent (1% trifluoroacetic acid in 80:20 (v/v) methanol:water) and transferred into autosampler vials for analysis.

Chromatography: Analytical separation was performed on the ExionLC AE system using a Luna omega polar PS C18 column (100 x 2.1 mm, 1.6 µm) at a 0.6 mL/min flow rate. Mobile phase A was 0.1% (v/v) formic acid in water and mobile phase B was acetonitrile. The column temperature was set to 75°C. The gradient conditions used are summarized in Table 1. A 10 μL sample was used for LC-MS/MS analysis.

image-bottom

Mass spectrometry: The optimized source and gas parameters are listed in Table 2 and the MRM parameters are included in Table 3.

image-bottom

image-bottom

Data processing: Analysis was performed using SCIEX OS software, version 3.4.5. Peaks were integrated using the MQ4 algorithm, and a weighting of 1/x² was used for pramlintide quantitation.

Quantitative performance

A triplicate injection was performed across concentrations ranging from 10 pg/mL to 100000 pg/mL (Figure 2). An LDR of 4 orders of magnitude was achieved. A weighing factor of 1/x2 was used with a coefficient of determination (r  ) of >0.994, showing excellent linearity across a wide calibration range.

Analytical performance was evaluated based on the requirement that the accuracy of the calculated mean should be between 80% and 120% at the LLOQ and between 85% and 115% at higher concentrations. The %CV of the calculated mean of the concentration should be below 20% at the LLOQ and below 15% at all higher concentrations.⁵

The accuracy was within ±10% of the nominal concentration and the %CV was <11 for the quantitation of pramlintide in human serum (Figure 3). Calculated %accuracy and %CV values were within the acceptance criteria at each concentration level.

Recovery was evaluated for pramlintide at 3 different concentrations (25, 40000 and 80000 pg/mL). A triplicate injection of the serum-extracted sample was compared against the post-spiked samples. An average recovery of 68.1% was achieved with a %CV <5.75 (Table 4).

Figure 2. Calibration curve for the quantitation of pramlintide in human serum with a weighting factor of 1/x². An LDR of 4 orders of magnitude was achieved with a r² >0.994.

image-top

Quantitative

Table 4. Recovery were evaluated using 25 pg/mL, 40000 pg/mL and 80000 pg/mL of pramlintide. Each concentration was evaluated in triplicate injections.

image-top

Figure 3. Quantitative performance for pramlintide (m/z 988.3 → m/z 968.0) analysis. Reproducibility and accuracy results were determined from the calibration curve standards across 3 replicates at each concentration. Statistical results were summarized using the Analytics module in SCIEX OS software.

image-top

Compliance

Compliance-ready SCIEX OS software

Equivalent SCIEX OS software capabilities for regulated bioanalysis can be executed on the SCIEX 7500+ system, ensuring high fidelity when performing method transfers while retaining critical compliance features. SCIEX OS software is a closed system and requires records and signatures to be stored electronically, meeting the regulations outlined by 21 CFR Part 11. SCIEX OS software can open raw data files from any visible storage location within a closed network by using designated processing workstations.

Figure 4 illustrates the features of SCIEX OS software used to monitor the audit trail, acquire and process data, and configure user access. The audit trail feature enables users to audit critical user actions and locks in data integrity. The Central Administrator Console (CAC) feature allows users to centralize acquisition and processing using a single platform to maximize efficiency for multi-instrument laboratories, independent of compliance standards. The configuration module allows users to assign roles and access as the administrator, method developer, analyst and reviewer.

Figure 4. Features of SCIEX OS software for monitoring user access and evaluating the audit trail. The audit trail view allows users to filter for high-risk events easily and enables data integrity features to meet compliance requirements. The software features a Central Administrator Console (CAC) to manage users and groups, role definitions, workstations and projects across all systems. The CAC feature supports both regulated and non-regulated compliance standards. The configuration module enables users to quickly set up roles and levels of access for the administrator, method developer, analyst and reviewer levels.

image-top

Conclusion

Conclusions

• An LLOQ of 10 pg/mL was achieved for quantitation of pramlintide in human serum
• Low-level quantitation was accomplished with 100 µL of human serum using a simplified SPE methodology
• Fast analysis of pramlinitide was performed using a 6-minute run time
• Linearity was achieved at concentrations ranging from 10 pg/mL to 100000 pg/mL, achieving an LDR of 4 orders of magnitude
• Good quantitative performance was demonstrated with accurate and highly reproducible (%CV <11) results on the SCIEX 7500+ system
• An average recovery of 68.1% with a %CV <5.75 was demonstrated for pramlintide analysis in human serum
• Retain data management and compliance-readiness (21 CFR Part 11) features using SCIEX OS software to support regulated bioanalysis on the SCIEX 7500+ system

References

References

1. Center for Drug Evaluation and Research, approval package for: Application number 21-332.
2. Bronislava R Gedulin, Andrew A Young. Hypoglycemia overrides amylin-mediated regulation of gastric emptying in rats. Diabetes. 1998;47: 93-97.
3. John Pullman, Tamara Darsow, Juan P Frias. Vasc Health and risk management. 2006;2(3):203-212.
4. R. G. Thompson, L. Pearson, O. G. Kolterman. Effects of 4 weeks' administration of pramlintide, a human amylin analogue, on glycaemia control in patients with IDDM: effects on plasma glucose profiles and serum fructosamine concentrations. 1997;40:1278-1285.
5. Bioanalytical Method Validation, May 2018