Evaluation Of Cartridge-Saturation versus OTC Drug Interference

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INTRODUCTION
Solid-Phase-Extraction (SPE) is a powerful tool for extraction of drugs from biological samples. It is reliable and efficient and it can be easily automated for high-throughput bioanalysis. SPE is particularly suitable in LC-MS/MS detection when high sensitivity is required because it leads to a clean extraction with minimized ionization suppression. With the use of new mixed-mode ion-exchange sorbents, the extraction of very-polar or ionized analytes, which are difficult to extract in Liquid-Liquid-
Extraction or on regular Reverse-Phase (RP) SPE cartridges, can be easily achieved obtaining high recovery and clean extracts.

However, it was found in this study that some of these sorbents are easily overloaded when an excess of competing analytes is present in the sample, which could produce uncontrolled variability.

MATERIAL AND METHODS

Sample Extraction
All calibrants and quality control (QC) samples were prepared in heparinized human plasma and spiked with Hydromorphone and Codeine as internal standard. Samples were buffered with a solution of ammonium acetate and applied to mixed-mode strong-cation-exchange SPE cartridges. Cartridges were washed with water and methanol and drugs were eluted with a methanolic ammonia solution. The eluates were evaporated to dryness and reconstituted in mobile phase.

Instrumentation
The quantitative analysis of Hydromorphone was performed using an Agilent Technologies Series 1100 pumps and autosampler coupled to an API 3000 tandem mass spectrometer (Applied Biosystems/MDS-SCIEX). Extracted sample aliquots were injected on an Atlantis Hilic Silica column (15 x 2.1 mm, 3µ, Waters).

The mobile phase consisted of acetonitrile: 0.1% acetic acid, 0.014% ammonium hydroxide in water (90:10% v/v) and the flow rate was set to 600 µL/min. The TIS source was set at 350°C and used in the positive ion mode. The following transitions were monitored for the detection of Hydromorphone and ISTD; m/z 286.2g185.1 m/z and 300.2g215.3, respectively.

RESULTS AND DISCUSSION

Hydromorphone Bioanalytical Method Development
A bioanalytical method was first developed to quantify the basic drug Hydromorphone in human plasma with a lower limit of quantification (LLOQ) of 25.0 pg/mL. To reach desired sensitivity for a reliable quantification, 1mL of plasma sample was
extracted by mixed-mode strong-cation-exchange SPE and reconstituted in a
final volume of 200µL of mobile phase. By doing so, the analytes were concentrated five times and adequate sensitivity was obtained. This assay showed good specificity and linearity (r2=0.9990).
Inter-run precision and accuracy ranged from 3.5-9.8% and 102.4-107.8%
respectively (Table I).

Presence of OTC Drugs Induces
Variations in Hydromorphone Quantification
To assess method selectivity over potential interferences that might come from different drugs present in plasma samples, 10 over-the-counter (OTC) drugs were spiked individually at twice their respective Cmax concentrations in low and high QC samples of Hydromorphone. These OTC spiked QCs were extracted according to the Hydromorphone method along with a Hydromorphone calibration curve and QCs, both free of OTC drugs.

The concentration of Hydromorphone in the OTC spiked QCs was evaluated from the Hydromorphone calibration curve. Deviation of the OTC spiked QCs back calculated concentrations from their nominal concentrations is represented in Table II. Significant variation can be observed for the majority of OTC spiked QCs. This variation is either the result of a drop in signal intensity of Hydromorphone, ISTD or both (raw data not shown). Interestingly, pronounced variation is observed from QCs that have been spiked with a basic OTC drug (see OTC Character in Table II).

This variation is caused by the presence of OTC drugs in the samples since the entire curve QCs, which are free of OTC drugs, show acceptable variation (Table II, Curve QC-1 to Curve QC-6). Moreover, no interfering peaks were detected at the transition of Hydromorphone or ISTD in extracted blank plasma samples spiked with OTC drugs (data not shown). Therefore, the variation observed in the OTC spiked QCs is either a result of an ionization suppression caused by the OTC drugs in the extracted sample or a competition between the analytes and the basic OTC drugs on the SPE cartridges.

Evaluation of Ionization Suppression Caused by OTC Drugs
To assess if the deviation caused by the OTC drugs is due to an ionization suppression effect, blank plasma and blank plasma spiked with OTC drugs were extracted and reconstituted with a pure solution of Hydromorphone and IS (corresponding to an extracted low QC or high QC concentration). The
ratio of Hydromorphone over ISTD of extracted blank and blank spiked with OTC drugs were compared (Table III). No significant deviations were observed, indicating that there was no ionization suppression caused by the presence of OTC drugs in the extracted samples. This shows that the variability comes from the extraction rather than the detection.

Evaluation of Cartridges Saturation Caused by OTC Drugs
Since most of the OTC drugs that induce variation in the quantification of Hydromorphone are basic drugs, it is possible that competition occurred for similar binding sites on the SPE sorbent between the basic drugs Hydromorphone and Codeine (ISTD), plasma interfering components and the excess of spiked OTC drugs. Therefore, the OTC interference evaluation was re-performed by using less overloading conditions. It was discovered that, at an amount of plasma equal to 0.5 mL, the saturation effect of the OTC drugs was overcome and all the OTC spiked QCs deviations were acceptable (Table IV). These results suggest that the cartridges were overloaded by using 1.0 mL of plasma.

A second method was therefore developed by reducing the amount of plasma used for the extraction. This second assay showed good specificity and linearity (r2=0.9997) over the range of 25.0 pg/mL—10,000.0 pg/mL. Inter-run precision and accuracy ranged from 4.7–9.6% and 98.5–100.9% respectively.

CONCLUSIONS

A precise and accurate bioanalytical method for Hydromorphone was developed using 1.0 mL of plasma as starting material. However, when using 1.0 mL of plasma, an excess of spiked OTC drugs leads to uncontrolled variability of Hydromorphone quantification in OTC spiked QCs. The fact that the majority of OTC drugs that produced significant deviation are basic drugs suggests that the strong-cation-exchange SPE cartridges were overloaded. The effect of the OTC drugs was overcome by using a smaller volume of plasma as starting material. These tests are performed systematically in the laboratory during ion-exchange-SPE method development to verify if SPE sorbents are saturated by competing OTC drugs present in plasma samples.