Simultaneous Determination of Sufentanil and Ziconotide in Combination for Intrathecal Analgesia by UPLC-UV

Introduction

Intrathecal analgesia has increased over the past two decades based on high level proof of efficacy in patients with cancer [1]. Morphine and Ziconotide are the only two agents approved by the US Food and Drug Administration (FDA) [2]. Morphine remains the reference in case of strong to intense pain [3]. Ziconotide is a small ω-conotoxine peptide isolated from a marine cone snail Conus magus [4]. It acts as a selective blocker of voltage-sensitive calcium channels of N-type [3–6]. Previous published studies demonstrated that the presence of morphine sulfate accelerated ziconotide degradation [7, 8].

Development of tolerance and side effects has forced clinicians to explore alternative opioids such as fentanyl and sufentanil [9]. The use of other drugs is common among clinicians: bupivacaine, ropivacaïne, clonidine, fentanyl, sufentanil [10].

Ziconotide/opioids (fentanyl, sufentanil) combinations was recommended by the 2012 Polyanalgesic Consensus Conference (PACC) IT treatment algorithm as the second and third line therapies in nociceptive pain and neuropathic pain respectively [10].

The dose used to start are for ziconotide from 0.25 to 0.50 µg/day [3] and for sufentanil 10 to 20 µg/day [10]. In clinical practice of our institution median concentration used for ziconotide is 0.58 µg/mL.

The administration of these analgesics drugs is done by using an intrathecal drug pump connected with a tunneled catheter implanted in the intrathecal area. This pump has a reservoir which must to be refill. This refill is prepared under strict aseptic conditions in a vertical laminar flow cabinet in a controlled atmosphere area. The two drugs are mixed together with a saline solution of 0.9 % as excipient (Fresenius Kabi, Sèvres, France) to achieve a final volume of 50 mL.

The preparation of the refill is performed using syringe 1, 5, 10, 20, and 50 mL syringes Plastipak (BD, Le Pont de Claix, France). No studies on the physicochemical stability of this mixture in a syringe were previously published. However a study on the stability in pump was published and shows a stability of 33 days [11]. Refills are prepared extemporaneously.

The prospective assay of Dupoiron et al. show the benefits of an analytical control to secure preparations and limits dosage errors [3].

The aim of this study was to develop and validate a new method of analytical control of sufentanil and ziconotide using ultra-performance liquid chromatography (UPLC) combined with an ultraviolet detector.

Materials and methods

Chromatographic and UV detection conditions

Analyses were conducted using an UPLC Acquity^® H-Class^® with UV detection (Waters, Guyancourt, France) managed by managed by the Empower software (V3) (Waters, Guyancourt, France). The column used was an Acquity UPLC^® BEH C18 1.7 µm 2.1×50 mm. The mobile phase was a gradient of ultrapure water with 0.1 % trifluoroacetic acid and acetonitrile with 0.1 % trifluoroacetic acid (Table 1). 10 µl were injected.

Table 1:

Gradient used for LC analysis.

Time (min)	Flow rate (mL/min)	% solvent A	% solvent B
0	0.5	95	5
0.5	0.5	90	10
0.9	0.5	75	25
2.0	0.5	75	25
3.5	0.5	55	45
4.8	0.5	0	100
5.8	0.5	0	100
6.1	0.5	95	5
8.5	0.5	95	5

Quantification was carried out at 200 nm for ziconotide and 220 nm for sufentanil. The retention times were of 1.69 min (Figure 1) and 3.85 min respectively (Figure 2).

Figure 1:

Chromatogram of Ziconotide.

Figure 2:

Chromatogram of Sufentanil.

Results

Discussion

The low concentrations of ziconotide associated with precision syringes are factors increasing the variability of results for ziconotide accuracy and precision.

After validation, this method has been used routinely. The acceptance criteria were ±10 % for each drug. When the error rate is above 10 % the preparation was homogenized again and analyzed. If the second result was above 10 %, the preparation was rejected. In two months, 250 preparations have been analyzed. Any preparation was rejected.

Conclusion

In order to secure the preparation, a new method based on UPLC-UV to quantify admixture with sufentanil and ziconotide was developed and fully validated according to FDA recommendations. The calibration range was from 0.1 to 4 μg/mL for ziconotide and 3.125 to 50 µg/mL for sufentanil. This method offers an interesting compromise in terms of detection level and analysis time compared with methods previously published. This routine quality control analysis secures the production process.