Stability study of hydromorphone and bupivacaine mixture by HPLC-UV

Abstract

Objectives

The objective of this study was to evaluate the physical and chemical stability of hydromorphone hydrochloride and bupivacaine hydrochloride in concentrations of 15 mg.ml^-1 and 10 mg.mL^-1 in 0.9% sodium chloride injection. Test samples of hydromorphone/bupivacaine mixtures were stored at 37°C, body temperature encounterd during continuous intrathecal infusion, for 90 days. The solutions were packaged in 20 ml plastic syringes. Evaluations for physical and chemical stability were performed initially and throughout the storage periods. Physical stability was assessed by visual observation. The chemical stability of the drug was evaluated by means of a stability-indicating high-performance liquid chromatographic (HPLC) analytical technique. In addition, pH and osmolarity were measured electronically.

Methods

This study determines the stability and compatibility of hydromorphone (15 mg.ml^-1) and bupivacaine (10 mg.ml^-1) mixture after 3 months at 37°C using a validated method by HPLC-UV. A simple, precise, specific and accurate reversed phase high performance liquid chromatographic (RP-HPLC) method was developed and validated. The different analytical performance parameters such as linearity, accuracy, specificity, precision and sensitivity (limit of detection and limit of quantitation) were determined according to International Conference on Harmonisation ICH Q2 (R1) guidelines. RP-HPLC was conducted on a nucleoshell RP18plus (C18 150×4.6 mm with 2.7 µm particle size) column. The mobile phase consisted of buffer A (phosphate buffer (0.05M) pH 4.5) and acetonitrile B. The gradient used for the elution is the following one: time (min)/% of B: 0 min/20%; 1.9 min/50%; 2.5 min /40%; 4.5 min/40%; 5.5 min/20%; and 8 min /20%, and the flow rate was maintained at 1.0ml.min⁻¹ and performed at 35°C. The molecules were monitored using Dionex ultimate 3000, equipped with photo diode array detector (λ=210 nm). Linearity was observed in concentration range of 9−21 mg.l^-1 for hydromorphone and 6-14 mg.l^-1 for bupivacaine. All the system suitability parameters were found within the range.

Results

The degradation study shows a photolytic degradation compound for hydromorphone and an oxidative degradation compound found for bupivacaine. The stability study shows no visible haze or particulate formation or gas evolution. pH and osmolarity were stable during the 3 months. Colour changed after 2 months, although this colouring is due to hydromorphone, proportional to hydromorphone concentrations and increases with time but it is a well known modification. The quantitative study by HPLC method revealed no significant change in hydromorphone and bupivacaine concentration. There is less than 5% of variability during the 3-month period.

Conclusions

Hydromorphone (15 mg.ml^-1) and bupivacaine (10 mg.ml^-1) were physically and chemically compatible and analysed with HPLC, which revealed no significant change in hydromorphone and bupivacaine concentration in this simulated compatibility study.

Objectives

Continuous intrathecal infusion of analgesic drugs by implantable pumps is recognised as an established treatment option for patients with chronic or neurologic pain.^1–4 For administration of analgesic drugs into cerebrospinal fluid, an implantable infusion system is introduced into the patient for approximately 5 years and the drugs solutions are changed every 3 months maximum. However, there are only a few published stability studies on intrathecal polyanalgesia under clinical conditions.⁵ Morphine is a reference for chronic pain by intrathecal infusion, but analgesic departments are using combinations of analgesic drugs as alternative treatments.^{2 6 7} The Fribourg Hospital Analgesic Department would like to know the compatibility and stability of high concentrations of hydromorphone/bupivacaine for intrathecal analgesia.

The main anaesthesic action of bupivacaine is exercised on myelinated nerve axons by a direct modification of Na⁺ channels. Hydromorphone is an opioid analgesic derived from morphine. It has a shorter duration of action and is more potent than morphine. Hydromorphone interacts predominantly with the opioid mu-receptors.

Research has been carried out on the intrathecal delivery of hydromorphone via implantable infusion pumps and has been demonstrated to be stable, safe and compatible with the delivery system.⁸ The same results for the stability, compatibility and safety of bupivacaine for intrathecal administration have been published.

There are no data regarding the stability and compatibility of high concentrations of hydromorphone/bupivacaine during intrathecal administration conditions. This study aims to determine the stability and compatibility of the hydromorphone (15 mg.ml^-1) and bupivacaine (10 mg.ml^-1) mixture after 3 months at 37°C using a validated method by HPLC-UV.

Methods

Materials

For the analytical method, hydromorphone hydrochloride was purchased from Macfarlan Smith (Edinburgh, Scotland) and bupivacaine hydrochloride from sigma Aldrich (St. Louis, MO, USA).

The solutions used for the syringes' preparations were purchased from Grossen Apotheke Dr. G. Bichsel AG (Interlaken, Switzerland) for hydromorphone hydrochloride 20 mg.ml^-1 and Sintetica (Mendrisio, Switzerland) for bupivacaine hydrochloride 4%.

HPLC-grade acetonitrile was purchased from Carlo Erba Reagents and potassium dihydrogenophosphate from Merck Millipore. The water used was purchased from Laboratorium Dr G. Bischel and the colour reference solution according to Ph. Eur. was purchased from Merck Millipore.

Equipment

The analysis was performed using a Dionex ultimate 3000, high performance liquid chromatography equipped with a diode array detector. The column employed was a nucleoshell RP18plus, C18 150×4.6 mm with 2.7 µm particle size, supplied by Macherey-Nagel. The system was piloted by chromeleon software 7.2.

The pH of the solutions was obtained using an Inolab level 3 pH/ion analyser. The osmolality of the solutions was obtained using an Advanced model 3300 osmometer.

The oven used to maintain the solution at 37°C was a Memmert oven SFP500.

Drug preparation

The drugs solutions employed in this study were prepared by the pharmacy department of Fribourg Hospital, Switzerland. Solutions of hydromorphone 15 mg.ml^-1 and bupivacaine 10 mg.ml^-1 were prepared under sterile conditions. Each preparation was verified by our validated analytical method to be within 5% of the stated concentrations.

The hydromorphone and bupivacaine mixture was conserved at 37°C and protected from light.

For the HPLC method, acetonitrile and phosphate buffer (0.05 M) pH 4.5 were used. Phosphate buffer was prepared using potassium dihydrogen phosphate (6.80 g in 1 L water). The buffer solution was filtered through a 0.45 µm filter (MF-Millipore Membrane Filter, 0.45 µm pore size).

Chromatographic condition

The buffer solution was prepared by dissolving 6.8 g of potassium dihydrogen phosphate in 1000 mL water. The injection volume and detection wavelength were fixed at 20 µL and 210 nm, respectively. The separation was employed using gradient elution with phosphate-buffer (mobile phase A) and acetonitrile (mobile phase B) based on the following programme: time (min)/mobile phase B(%): 0/20; 1.9/50; 2.5/40; 4.5/40; 5.5/20; 8/20.

All separations were performed at 35°C and using a flow rate of 1 mL.min^-1. Under these conditions, hydromorphone retention time was 1.81 min ±0.01 and bupivacaine retention time was 3.91 min ±0.16.

Method’s validation

This method was developed and validated using ICH guidelines and SFSTP recommendations.^{9 10}

Linearity

The linearity of the method was established by triplicate injections in the range of 9–21 mg.l^-1 for hydromorphone and 6–14 mg.l^-1 for bupivacaine corresponding to 80, 90, 100, 110and 120% of the nominal analytical concentration. The linearity was evaluated by linear regression analysis, which was calculated by the least-square regression analysis.

Specificity

The specificity of the developed HPLC-UV method for the quantification was investigated by chromatographic analysis of each compound independently and of the degradation studies.

Degradation study

Hydromorphone and bupivacaine solutions were prepared at concentrations of 60 mg.l^-1 for hydromorphone and 40 mg.l^-1 for bupivacaine.¹¹ Solutions were stressed by different conditions:

• Solutions were stocked with 50 µL H₂O₂3% and 30% for 1000 µl of solution.

• pH of solutions were adjusted at PH=3 with HCl concentrated and PH=9 with NaOH concentrated.

• Solutions were placed at 85°C.

• Solutions were exposed under UV light.

Precision

Precision of the method was determined by repeatability (intraday precision) and intermediate precision (interday precision). Results are presented as relative standard deviation (RSD %).¹²

For interday precision, the study was repeated on three different days. For intraday precision, six solutions in the middle of the range (15 mg.l^-1 for hydromorphone and 10 mg.l^-1 for bupivacaine) were analysed by independent weights and injected for each day. The recoveries were calculated.

Each solution was made with independent weights of hydromorphone and bupivacaine.

The resolution factor of hydromorphone from bupivacaine was also determined. Limits of detection and limits of quantification were determined for each compound.

Sensitivity

Limit of detection (LOD) and limit of quantitation (LOQ) were determined using the calibration curve method according to ICH Q2 (R1) recommendations. The LOD (k=3.3) and LOQ (k=10) of the proposed method were calculated using the following equation:

A=kσ/S, where A is LOD or LOQ, σ is the SD of the response, and S is the slope of the calibration curve.

Results

Method’s validation

Linearity

The regression equation for hydromorphone and bupivacaine, is defined by plotting peak area (y) versus the concentration (x) studied from, and the correlation coefficient (R ²>0.99). The validity of the assay was verified by means of the ANOVA. According to it, there is linear regression and there is no deviation from linearity (P<0.05). The model is linear.

Specificity

A typical HPLC-UV chromatogram of hydromorphone standard preparation and bupivacaine test sample is shown in figure 1. The retention times recorded for each compound are checked.

Figure 1.

Bupivacaine chromatogram solution at 10 mg.l^-1 and hydromorphone chromatogram at concentration 15 mg.l^-1.

Degradation study

A photolytic degradation compound was found for hydromorphone and an oxidative degradation compound for bupivacaine. The results are detailed in table 1. The retention times are 3.91 min and 4.40 min, respectively, for bupivacaine and degradation product. The retention times are 1.91 min and 1.40 and 1.72 min, respectively, for hydromorphone and degradation products. The presence of interfering peak(s) eluted at/or near the retention time of hydromorphone and bupivacaine was also checked. The forced degradation study showed no interference with degradation products.

Table 1.

Results of hydromorphone and bupivacaine exposed to different degradative pathways

Parameter	Percentage of degradation of hydromorphone	Percentage of degradation of bupivacaine	Exposition (days)
PH=3	<3%	<3%	19
PH=9	<3%	<3%	19
H2O2 30%	45%	64%	19
H2O2 3%	31%	60%	19
Photolytic	37%	<3%	19
85°C	<3%	<3%	4,5

Accuracy

The overall results of recoveries (mean ± %RSD) of hydromorphone and bupivacaine are detailed in table 2. The 95% trust intervals including covering rate 100% are detailed in table 3. These results are indicating good accuracy of the proposed HPLC-UV method.

Table 2.

Accuracy studies of hydromorphone and bupivacaine

Hydromorphone				Bupivacaine
Amount added (µg.mL−1)	Amount recovered (µg.mL−1)	Recovery %	Recovery (mean ± %RSD)	Amount added (µg.mL−1)	Amount recovered (µg.mL−1)	Recovery %	Recovery (mean ± %RSD)
8.97	9.07	101.15	100.51±0.01	6.07	6.17	101.67	101.59±0.02
9.09	9.03	99.37		6.04	6.26	103.57
9.00	9.09	101.00		5.97	5.94	99.53
12.08	12.01	99.43	99.39±0.01	7.88	7.82	99.16	98.97±0.01
11.96	11.80	98.62		8.05	7.98	99.06
12.12	12.13	100.11		7.88	7.78	98.70
14.85	14.86	100.06	99.71±0.01	9.95	9.85	99.04	98.81±0.01
15.25	15.15	99.35		9.93	9.87	99.48
14.95	14.91	99.72		9.95	9.74	97.92
17.94	17.83	99.37	100.70±0.012	11.82	11.95	101.07	100.83±0.01
18.00	18.08	100.44		12.00	11.94	99.56
18.12	18.53	102.29		11.97	12.19	101.87
21.21	21.04	99.22	99.75±0.01	13.90	13.89	99.94	100.03±0.01
21.14	21.11	99.84		14.06	13.94	99.17
20.86	20.90	100.18		13.99	14.13	100.99
9.06	9.45	104.29	100.05±0.04	6.03	6.13	101.68	100.61±0.01
9.06	8.79	97.04		6.03	6.05	100.40
9.09	8.98	98.82		6.03	6.01	99.75
11.92	11.74	98.48	100.12±0.02	8.07	8.04	99.65	100.34±0.01
12.00	12.23	101.90		8.00	8.02	100.30
12.00	12.00	99.98		7.92	8.00	101.06
14.9	14.84	99.58	99.49±0.01	9.95	9.96	100.08	99.61±0.01
15.15	15.10	99.66		9.93	9.86	99.33
15.1	14.98	99.22		10.04	9.99	99.44
18.12	18.21	100.52	100.50±0.01	12.05	11.76	97.59	98.67±0.02
17.94	18.12	101.00		11.82	11.94	101.00
18.18	18.17	99.97		12.08	11.77	97.44
21.21	21.17	99.82	99.85±0.01	14.06	14.13	100.47	100.97±0.01
20.86	20.87	100.03		13.96	14.22	101.82
21.14	21.07	99.69		14.03	14.12	100.63
9.06	8.99	99.26	99.29±0.01	6.03	6.43	106.72	100.22±0.06
9.00	8.94	99.33		5.96	5.74	96.32
9.00	8.94	99.29		6.03	5.88	97.62
12.04	12.01	99.78	100.28±0.01	7.98	8.33	104.46	100.11±0.04
12.12	12.06	99.50		7.94	7.63	96.16
11.92	12.11	101.57		7.94	7.92	99.73
14.90	14.80	99.30	99.90±0.01	10.07	10.40	103.27	100.50±0.02
15.15	15.26	100.72		10.09	9.97	98.85
15.05	15.00	99.67		10.00	9.93	99.38
18.24	18.12	99.34	100.96±0.02	11.91	11.84	99.43	98.57±0.01
17.94	18.35	102.28		12.05	11.76	97.61
17.94	18.16	101.24		11.97	11.81	98.67
21.00	20.74	98.78	99.41±0.02	14.09	14.38	102.05	100.69±0.02
21.14	20.79	98.33		14.03	13.74	97.93
20.93	21.17	101.13		14.13	14.42	102.07

Table 3.

Trust intervals 95% for hydromorphone and bupivaine

Days Results	1	2	3
Trust interval 95% for hydromorphone	99.59<C<100.43	99.26<C<100.74	99.39<C<100.45
Trust interval 95% for bupivacaine	99.40<C<100.75	99.47<C<100.62	98.62<C<101.42

Precision

The values of %RSD for intraday and interday variations are detailed in table 4. In both cases, the RSD values obtained were always lower than 5%, meaning that the current method is repeatable.

Table 4.

Precision of HPLC method for hydromorphone and bupivacaine

Day	Sr. no.	Hydromorphone 15 µg.mL−1		Bupivacaine 10 µg.mL−1
		Peak area	Intra-day precision	Peak area	Intra-day precision
1	1	21.45	Mean:21.52 SD:0.33 %RSD:1.55	9.70	Mean:9.59 SD:0.11 %RSD:1.18
	2	21.86		9.72
	3	21.52		9.59
	4	21.34		9.86
	5	22.04		9.73
	6	22.14		9.90
2	1	21.69	Mean:22.02 SD:0.30 %RSD:1.38	9.82	Mean:9.93 SD:0.15 %RSD:1.54
	2	22.06		9.72
	3	21.89		9.86
	4	22.11		10.10
	5	21.80		9.96
	6	22.55		10.09
3	1	21.46	Mean:21.98 SD:0.37 %RSD:1.68	10.43	Mean:10.29 SD:0.24 %RSD:2.33
	2	22.12		10.02
	3	21.75		9.98
	4	21.94		10.39
	5	22.56		10.35
	6	22.02		10.58
Inter-day precision	Mean	21.91		9.99
	SD	0.34		0.28
	%RSD	1.57		2.85

Sensitivity

The LOD and LOQ of hydromorphone by the proposed method were equal to 1.91 mg.mL^{− 1} and 5.80 mg.mL^{− 1}, respectively. The LOD and LOQ of bupivacaine by the proposed method were equal to 1.61 mg.mL^{− 1} and 4.89 mg.mL^{− 1}, respectively.

Syringe analysis

The results are presented in the table below.

The results of the stability study for the mixture solution hydromorphone (15 mg.ml-1) and bupivacaine (10 mg.ml-1) are detailed in table 5. The quantitative study by HPLC method, revealed no significant change in hydromorphone and bupivacaine concentration. There is less than 5% of variability during the 3-month period. Limpidity was correct, there are not suspended particles. pH and osmolarity were stable. The colour changed after 2 months, although this colouring is proportional to hydromorphone concentration and increases with time – it is a well known modification.^{13 14} The solution will be injected by intrathecal pump.

Table 5.

Results of mixture analysis

Analysis	Month
	0	1	2	3
Limpidity	Not suspended particles	Not suspended particles	Not suspended particles	Not suspended particles
Concentrations
Hydromorphone	103.91%	102.49%	101.70%	101.91%
Bupivacaine	99.48%	101.01%	98.65%	96.54%
pH	5.10	5.66	5.71	5.67
Osmolarity	285.0	289.2	291.1	292.8
Degree of c olouration	Less coloured than control B6*	Less coloured than control B6*	Less coloured than control B6* and no more coloured than control B5*	Less coloured than control B5* and no more coloured than control B4*

Conclusion

Drug combinations provide several advantages for long-term intrathecal pain management. Chemical and physical compatibility of drug mixture injected by intrathecal pump should be studied to certify the mixture’s compatibility. During this study, the HPLC-UV method was developed and validated to analyse and determine the stability of the hydromorphone and bupivacaine mixture. ICH texts were used to validate this method and found a linearity, accuracy and repeatability concentration range.

The mixture study was hydromorphone (15 mg.ml^-1) and bupivacaine (10 mg.ml⁻¹). Long-term stability of the commonly used analgesic mixture of morphine sulphate, bupivacaine hydrochloride and clonidine hydrochloride in the SynchroMed infusion systems or syringe has been previously reported.^15–19 After 3 months at 37°C, the hydromorphone and bupivacaine mixture was stable in our conditions, except the colouring change, but was proven by other studies and there are no influences on stability.

What this paper adds.

What is already known on this subject

• The solution with 50, 24 and 2mg.ml^-1 for hydromorphone hydrochloride, bupivacaine hydrochloride and clonidine hydrochloride, respectively, is stable at 37°C during 90 days (F. Bianchi, A. Ginggen and Y. Tardy)

• Bupivacaine HCl (0.25%) and hydromorphone (0.02 or 0.04 mg/mL) stored at either 22°C or 6°C, packaged in polypropylene syringes, were considered stable for at least 91 days. (Ronald Donnelly. The Ottawa Hospital)

What this study adds

• A validated method by HPLC-UV/DAD with a degradation study for hydromorphone and bupivacaine

• The hydromorphone (15 mg.ml^-1) and bupivacaine (10 mg.ml^-1) mixture is stable after 3 months at 37°C.