Research Article
`LIQUID CHROMATOGRAPHY-MASS SPECTROMETRY METHOD TO ASSESS NALOXONE
`HYDROCHLORIDE PHOTOSTABILITY UNDER ARTIFICIAL LIGHT AND SUNLIGHT EXPOSURE
`AT ROOM TEMPERATURE
`
`NASER F ALTANNAK*
`Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Kuwait University, P.O Box 23924, Safat, 13110 Kuwait.
`Email: Dr_altannak@hsc.edu.kw
`Received: 27 June 2015, Revised and Accepted: 03 August 2015
`
`ABSTRACT
`
`INTRODUCTION
`
`Objectives: Naloxone is an opioid antagonist indicated for central nervous system and respiratory depression treatment induced by natural or
`synthetic opioid in adults and neonates whose mothers have received opioids. Although naloxone hydrochloride has been reported to be physically
`and chemically stable, photostability of naloxone hydrochloride under artificial light, and sunlight has not been reported. Therefore, a method was
`required for assessment of naloxone hydrochloride photostability.
`Methods: A high-performance liquid chromatography/mass spectrometry method was established to evaluate the photostability of naloxone
`hydrochloride. Injections of naloxone hydrochloride in 0.9% sodium chloride were exposed to artificial light and sunlight at room temperature for
`192 hrs.
`Results: Naloxone losses up to 5.26% of its initial concentration when exposed to artificial light at room temperature for 192 hrs, but the degradation
`increased up to 15.08% under sunlight exposure at room temperature for 192 hrs. The disappearance of naloxone hydrochloride was correlated with
`the appearance of noroxymorphone degradant.
`Conclusion: Naloxone hydrochloride is photosensitive and degradation increased as the light intensity increased. Therefore, naloxone intravenous
`infusion solutions should either be protected from light and/or be frequently replaced when being administered to patients.
`Keywords: Liquid chromatography/mass spectrometry, Naloxone hydrochloride, Photostability, Noroxymorphone, Intravenous infusion, Electrospray
`ionization.
`hydrochloride stability beyond 24 hrs in case of continuous occurrence
`of opioid symptoms [20,21]. Moreover, most of the designed studies
`Different classes of drugs have been developed to control different
`have used complicated and sensitive detectors which are not regularly
`types of pain, nevertheless none of these classes has replaced or
`used in analytical laboratories [22,23]. For example, one of the reported
`compete with opioid analgesics to manage moderate to severe
`methods involves the use of electrochemical detectors, which is highly
`pain [1-4]. Therefore, opioids are the first line treatment for cancer
`sensitive to the quality of water used in the mobile phase [24]. Although
`and postoperative pain. Unfortunately, opioid analgesics have serious
`naloxone hydrochloride is known to be a photosensitive compound, the
`possible side effects such as constipation, nausea, vomiting, drowsiness,
`previously reported literatures investigated only the effects of heat and
`and respiratory depression [5-12]. Thus, an opioid antagonist know
`humidity as the main factors affecting naloxone hydrochloride infusion
`as naloxone hydrochloride (N-allyl-noroxymorphone) is indicated for
`stability [16,20,21,25,26]. Thus, a simple stability indicating method
`complete or partial reversal of central nervous system and especially
`was designed to investigate the effect of artificial light and sunlight
`respiratory depression induced by over-dosage or intoxication of
`exposure on naloxone hydrochloride in 0.9% sodium chloride at room
`natural or synthetic opioids. Moreover, it can reverse the dysphoric,
`temperature for 192 hrs.
`delusional, and hallucinatory properties of synthetic opioid [13,14]. In
`addition, naloxone hydrochloride has been successfully administered
`by different routes. However, due to rapid onset and dose titration,
`the intravenous (i.v.) route seems to be the ideal route for naloxone
`hydrochloride administration [15,16]. Therefore, continuous infusion
`Naloxone hydrochloride (0.4 mg/ml) ampoules were a purchased
`of naloxone hydrochloride is recommended for neonate, whose
`from HIKMA Pharmaceutical (Amman). Isotonic sodium chloride
`mothers have received opioids, to treat respiratory depression and the
`solution for injection was supplied by Kuwait Saudi Pharmaceutical
`infusion can be continued for 2-5 days if the respiratory depression
`Industry Company (Kuwait). Naloxone hydrochloride dehydrate and
`recurred [17,18]. However, five impurities have been indicated
`high-performance liquid chromatography (HPLC) grade water were
`for naloxone hydrochloride by British Pharmacopeia
`including,
`purchased from Sigma-Aldrich (St. Louis, MO). Acetonitrile HPLC grade
`noroxymorphone, which is the starting material for the synthesis of
`was supplied by LiChroslov (Darmstadt, Germany). Formic acid (99-
`naloxone hydrochloride [19]. Noroxymorphone may cause nausea,
`100%) purchased from by Surechem Products Ltd. (16 Maitland Road,
`vomiting, abdominal pain, and constipation [20]. Therefore, naloxone
`Ipswich, England). Plastic syringes were purchased from Sensecure
`hydrochloride photostability should be indicated and the levels of
`(Loughborough, England).
`noroxymorphone present should be controlled. Although several
`methods are reported in the literature for the determination of
`The HPLC system (Waters 2690 Separation Module) used in this
`naloxone hydrochloride concentration in blood, urine and solution
`samples, few studies have been established to evaluate naloxone
`analytical method consisted of a Waters 600E multisolvent delivery
`
`METHODS
`
`Chemical and materials
`
`Instrumentation
`
`Vol 8, Issue 5, 2015
`
`ISSN - 0974-2441
`
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`Liquid chromatography-mass spectrometry (LC-MS)
`
`Time
`(hrs)
`
`Naloxone hydrochloride
`% Remaining
`% Remaining
`syringe 1
`syringe 2
`
`100
`98.54
`96.88
`96.58
`96.34
`94.74
`
`100
`0
`98.59
`4
`97.81
`24
`97.75
`48
`96.70
`96
`95.83
`192
`RSD: Relative standard deviation
`
`system pump, a Waters Ultra WISP 715 auto-injector, and a Waters
`996 diode-array detection system. Chromatographic separation was
`performed using Waters Symmetry, C-18 (5 µm, 150 mm × 4.6 mm i.d)
`column.
`Tandem MS was performed by a Waters Alliance 2695 Separations
`Module HPLC, equipped with a quaternary pump and an automatic
`interfaced to a Micromass Quattro micro API (triple quadrupole) MS
`equipped with a Z-spray electrospray (ESI) ionization source was
`used. Nitrogen as drying, as well as nebulizing gas, was generated
`from pressurized air in a NG-7 nitrogen generator. The nebulizing gas
`flow was set to 50 L/h and the gas flow desolvation to 550 L/h. The
`optimized values were: Capillary voltages, 4.5 kV; extractor voltage,
`2 V; source temperature, 100°C; desolvation temperature, 400°C; and
`multiplier, 650 V.
`The pH of the injection was measured prior to sample analysis with a
`pH meter that was calibrated with buffers at pH 4 and 7.
`Mobile phase comprised filtered and degassed 0.04% v/v formic acid in
`water and acetonitrile in proportion of 65:35 v/v and pumped at a flow
`rate of 1 ml/minutes. Samples were analyzed at a wavelength of 260 nm
`and were injected at 10 µm injection volume. In LC-MS, the same
`conditions as were used in HPLC but at a flow rate of 2 ml/minutes. LC
`was directly attached to the triple quadrupole MS via ESI.
`A stock solution containing 1 mg/ml of naloxone hydrochloride
`dehydrate dissolved in 0.9% sodium chloride solution was prepared
`and the linearity of response around the nominal content in the
`injection was achieved with six concentrations ranging from 25% to
`200% diluted with 0.0.4% formic acid in water:acetonitrile (65:35 v/v).
`Duplicate injections composed 0.2 mg/ml naloxone hydrochloride
`in 0.9% sodium chloride solution were prepared and stored at 22°C
`under artificial light and sunlight exposure. Samples were analyzed at
`0 minutes, 24, 48, 96, and 192 hrs.
`Figs. 1 and 2 shows the chromatograms obtained for naloxone
`hydrochloride after sunlight exposure for 24 hrs and 4 days, respectively.
`After 192 hrs under room temperature, injection solutions exposed to
`artificial light were prone to degradation up to 5.26%, while under
`sunlight exposure naloxone hydrochloride was degraded up to 15.08%.
`Tables 1 and 2 shows the set of data obtained for naloxone hydrochloride
`exposed to artificial light and sunlight, respectively. There was a loss of
`naloxone hydrochloride in the sample with a corresponding increase in
`the degradant peaks. Thus, MS was used to identify the degradant peak.
`Fig. 3 shows a full scan ESI MS of the degradant peak obtained in Fig. 2.
`The calibration curve for naloxone hydrochloride was linear over the
`range between 25% and 150% of the stated content for naloxone
`hydrochloride in 0.9% sodium chloride infusion with R2 of 1.00. Method
`precision was tested by preparing 10 samples containing 0.2 mg/ml
`of naloxone hydrochloride and analyzed using previously mentioned
`chromatographic conditions. The relative standard deviation of peak
`area obtained for naloxone hydrochloride was ±0.9%. Degradation of
`naloxone hydrochloride was noticeable under artificial light and sunlight
`exposure at room temperature. Under the examined conditions, one
`degradant peak is clearly eluted before naloxone hydrochloride peak.
`Noroxymorphone peak is formed and it becomes more prominent after
`48 hrs at room temperature under artificial light and sunlight exposure.
`As shown in Fig. 3, identification of degradant to be nornaloxone
`(noroxymorphone) [19,20] was performed using tandem MS. Visual
`inspection of naloxone hydrochloride injection solution showed that
`
`pH measurement
`
`Chromatographic conditions
`
`Sample preparation
`
`RESULTS
`
`DISCUSSION
`
`
`
`Altannak
`
`Asian J Pharm Clin Res, Vol 8, Issue 5, 2015, 309-311
`
`Table 1: Stability data obtained for naloxone hydrochloride
`(0.2 mg/ml) in in 0.9% sodium chloride under artificial light
`exposure at room temperature for 192 hrs
`
`RSD%
`
`0
`±0.03
`±0.7
`±0.9
`±0.3
`±0.8
`
`RSD%
`
`±0
`±2.7
`±0.6
`±0.1
`±0.3
`±0.6
`
`Table 2: Stability data obtained for naloxone hydrochloride
`(0.2 mg/ml) in in 0.9% sodium chloride under sunlight
`exposure at room temperature for 192 hrs
`
`Naloxone hydrochloride
`% Remaining
`% Remaining
`syringe 1
`syringe 2
`
`Time
`(hrs)
`
`0
`4
`24
`48
`96
`192
`
`100
`98.16
`88.04
`87.01
`86.01
`85.68
`
`100
`94.43
`88.73
`86.89
`85.71
`84.91
`
`Fig. 1: High-performance liquid chromatography analysis of
`0.2 mg/ml naloxone hydrochloride in 0.9% sodium chloride after
`24 hrs of sunlight exposure at room temperature
`
`Fig. 2: High-performance liquid chromatography analysis of
`0.2 mg/ml naloxone hydrochloride in 0.9% sodium chloride after
`4 days of sunlight exposure at room temperature
`
`the appearance remain unchanged for 192 hrs under artificial light
`exposure at room temperature (22°C), while a noticeable color change
`from colorless solution once prepared to a yellow-brownish solution
`310
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`
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`Altannak
`
`Asian J Pharm Clin Res, Vol 8, Issue 5, 2015, 309-311
`
`Fig. 3: Full scan electrospray ionization mass spectrum of
`noroxymorphone
`
`was performed due to extensive degradation after 7 days sunlight
`exposure at room temperature. Apart from the color change, the pH of
`naloxone hydrochloride injection solution remains stable over 192 hrs,
`and there was no evidence of particulate formation.
`The naloxone hydrochloride shown in the current study is considerably
`stable under artificial light exposure at room temperature (22°C) for
`192 hrs, while it shows more degradation under sunlight light exposure
`at room temperature. Moreover, the study showed that naloxone
`hydrochloride degradation is directly proportional to the amount of
`light exposed to it. Thus, naloxone hydrochloride i.v. infusion should be
`administered in a controlled light radiation and the infusion preferably
`changed if the infusion continued for more than 24 hrs.
`This work was supported by the Research Sector, Kuwait University
`Research Grant ZP03/13. Sincere gratitude is hereby extended to Ph.
`Vidhya Thomas for her technical assistance, Ph. Fatimah Al Shammari
`the chairman for injections department at Kuwait medical store for her
`help to supply us with naloxone hydrochloride ampules, my family, and
`finally to Prof. Oludotun A. Phillips and Prof. Mohammed Abdul-Hamid
`for their support and encouragement.
`
`CONCLUSION
`
`ACKNOWLEDGMENT
`
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