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Development and validation of UPLC/ESI-Q-TOF-MS for carteolol in aqueous humour: Stability, stress degradation and application in pharmacokinetics of nanoformulation
⁎Corresponding author. Mobile: +91 9899571726. alipharm@gmail.com (Asgar Ali)
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Received: ,
Accepted: ,
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.
Peer review under responsibility of King Saud University.
Abstract
Carteolol (CRT) is currently under development as a potential therapeutic agent for the treatment of open angle glaucoma. The purpose of the present work is to develop and validate a stability indicating assay method and its application to estimate CRT in aqueous humour and study the pharmacokinetic parameters. An ultra performance liquid chromatographic tandem mass spectroscopy (UPLC–MS/MS) method was developed and validated for the quantitative determination of CRT in rabbit aqueous humour, using propranolol as the internal standard (I.S.). Aqueous humour samples were prepared by a simple liquid–liquid extraction technique (LLE). The analyte and internal standard were separated by an Acquity UPLC BEH C18 (100.0 × 2.1 mm; 1.7 μm) column with a mobile phase of acetonitrile – 2 mM (milli mole) ammonium acetate (90/10, v/v) over 3 min of retention time. Detection was based on the multiple reactions monitoring with the precursor-to-product ion transitions m/z 293.2 → 237.12 for CRT and m/z 260.09 → 183.04 for I.S. The method was validated according to FDA guidelines on the bio-analytical method validation. The method developed was linear (r2 = 0.999) over the concentration range of 1–1000 ng/mL. The selectivity, sensitivity, linearity, accuracy, precision, extraction recovery, and stability were within the acceptable ranges. Forced degradation studies were performed on bulk sample of CRT as per ICH prescribed stress conditions, such as acid, base, oxidative and photolytic to show the forced of the method. Significant degradation was observed during basic stress condition. The pharmacokinetic study of CRT solution and nanoparticles in aqueous humour of rabbit eye was performed and results showed that CRT nanoparticles enhance the ocular bioavailability by 5.61-fold as compared to CRT-solution.
Keywords
Glaucoma
Carteolol
UPLC/ESI-Q-TOF-MS
Chitosan nanoparticles
Stress degradation
Pharmacokinetics
1 Introduction
Glaucoma is a group of diseases characterised by a progressive form of optic nerve damage associated with raised (>21 mmHg) intraocular tension (i.o.t) and reduction of the visual field. Many of drugs, including β-adrenergic blocking agents (β-blockers oral and eye drops), have been topically applied to lower intraocular pressure (IOP). Some of them have been prescribed concomitantly with the other antiglaucoma drugs, such as prostaglandins, acting by different mechanism in order to cause synergism (Zimmerman, 1993; Novack et al., 2002; Ohtake et al., 2004; De Santis, 1994). In spite of the wide use for glaucoma therapy, it is well known that topically administered β-blockers may cause severe cardiovascular side-effects, and nonselective β-blockers may cause respiratory failure (Shields, 1992). CRT is the second most commonly prescribed topical β-blocker in the world with intrinsic sympathomimetic activity and exert a partial agonism at the adrenergic receptor while simultaneously block natural endogenous agonist from binding to the receptor. This activity has proved effective in a real reduction of some side effects (Black and Mann, 1984; Moroi and Lichter, 2001; Nathanson, 1980). The chemical name of CRT is (±)-5-[3-[(1,1-dimethylethyl) amino]-2 hydroxypropoxy]-3,4-dihydro-2(1H)-quinolinone (Fig. 1A), available as 1–2% w/v aqueous eye drop solution, (Kuwahara et al., 2005; Trinquand et al., 2003). Almost all drugs administered as eye drops are drained from the precorneal area into the systemic circulation by the way of the nasolacrimal duct. Therefore the ocular bioavailability of drugs instilled is often very low, typically 1% or less (Jarvinen et al., 2000). This is one of the reasons causing such adverse effects of ophthalmic drugs. Various novel drug delivery systems such as in situ gelling system (El-Kamel et al., 2006), ion transport system (Higashiyama et al., 2006) and algenic acid (Séchoy et al., 2000) delivery have been investigated by many researchers in an effort to overcome poor bioavailability of CRT after topical application. So advantages of these novel formulations include prolonged pre-corneal retention and reduce the systemic side effect, which, in turn, is good for the quality of life of patients.
Quantification of CRT in biological fluid by chromatographic procedures like mass spectrometry (Zammataro et al., 2010) and HPLC (El-Kamel et al., 2006) has been reported previously. Quantification of CRT concentration in aqueous humour is described in the literature by HPLC (Zammataro et al., 2010). The combination of HPLC with the tandem mass spectrometer (MS/MS) has become more and more attractive for the quantification of drugs at low level (Plumb et al., 2004; Wood et al., 2006). This technique has been used to quantify CRT and dorzolamide simultaneously (Zammataro et al., 2010). As Compared to conventional HPLC analysis, ultra performance liquid chromatography (UPLC) performed with Acquity BEH 1.7 μm columns can provide greater peak capacity, higher speed and efficiency (De Villiers et al., 2006). Moreover, in comparison with HPLC–MS/MS, UPLC is a new chromatographic technique utilising high linear velocities, which is based on concept using columns with smaller packing (1.7–1.8 μm porous particles) and operated under high pressure (up to 15,000 psi). This is an extremely powerful approach which dramatically improves peak resolution, sensitivity and speed of analysis. In addition, time of-flight mass spectrometry (Q-TOF-MS) allows the generation of mass information with higher accuracy and precision. UPLC is specially designed to resist higher back-pressures, with the advantages of fast injection cycles, low injection volumes, negligible carryover and temperature control (4–40 °C), which collectively contribute to speedy and sensitive analysis. The UPLC–MS/MS technique produces significant improvements in method sensitivity, speed, and resolution (Abdelbary and Nebsen, 2013; Churchwell et al., 2005; Du et al., 2013; Gupta et al., 2013; Mazzarino et al., 2008; Nzoughet et al., 2013; Pedraglio et al., 2007; Thöngren et al., 2008; Wilson et al., 2005). Therefore, in this study, we developed a rapid, sensitive and selective UPLC–MS/MS method to quantify CRT in small volumes of aqueous humour and also do stress study the samples as per ICH guidelines (ICH. Topic Q2-R1, 2005). Liquid–liquid phase extraction was chosen to extract CRT from aqueous humour samples which resulted in good recovery, lack of endogenous interference, and moderate matrix effect. The proposed UPLC–MS/MS method with simple pre-treatment procedures was successfully applied to assess the pharmacokinetic profiles of CRT- solution and CRT nanoparticle formulation in aqueous humour of rabbit’s eye.
2 Experimental
2.1 Chemicals and reagents
CRT (purity: 99.98%) was a kind gift from Otsuka Pharmaceutical Co., Ltd, Japan. Chitosan (CS, deacetylation degree >85%) was received as a gift sample from India Sea Foods (Kerala, India). Acetonitrile and methanol were obtained from SD Fine (Mumbai, India). Ammonium acetate (∗∗LCMS grade), formic acid (LCMS grade), ammonium format and ethyl acetate were obtained from SD Fine (Mumbai, India). All solvents and reagents were of analytical reagent grade. Ultra-pure water was produced by a Milli Q (Millipore, Billerica, MA, USA) system. Eppendorf tubes (1–2 mL) were used to store solution during the preparation of the standard solution and aqueous humour samples.
2.2 Liquid chromatography
UPLC analysis of CRT in aqueous humour was performed on a Waters ACQUITY UPLC system (Waters Corp., MA, USA) equipped with a binary solvent delivery system, an auto-sampler, column manager and a tunable synapt MS detector (Synapt; Waters, UK). UPLC Chromatographic separation was performed on a Waters Acquity UPLC BEH C18 column (100 × 2.1 mm, 1.7 μm particle size) with Van Guard pre-column (5 × 2.1 mm, 1.7 μm particle size) waters acquity. The mobile phase consisted of acetonitrile: 2 mmol ammonium acetate buffer (90:10, v/v) at column temperature of 40 °C, auto sampler temperature at 4 °C and flow rate of 0.2 mL/min. The injection volume was 10 μl in full loop mode. Retention time was 1.29 min and total chromatographic run time was 3.0 min.
2.3 Mass spectrometry
Synapt-mass-spectrometric detection was carried out on a UPLC–MS/MS (Q-TOF-ESI) (Waters Corp., USA) with an electrospray-ionisation (ESI) technique. The ESI source was used in positive ionisation mode. Quantification was performed using synapt MS (Q-TOF) of the transitions of m/z 283.2 → 237.12 for CRT and m/z 260.09 → 183.04 for IS, with scan time of 1.0 min per transition and internal scan time was 0.05 s. Microchannel plate (MCP) was used as a detector plate. The optimal MS parameters are as follows: capillary voltage 3.60 kV, cone voltage 21 V, extraction cone voltage 7.40 V, source temperature 120 °C, desolvation temperature, 350 °C; cone gas flow, 50 l/h; desolvation gas flow, 500 l/h; and collision energy was 16.5. Nitrogen was used as the desolvation and cone gas. Argon was used as the collision gas at flow of 1.5 mL/min. All data collected in centroid mode were acquired and processed using MassLynx™ V 4.1 software.
2.4 Standard solutions, spiked samples and quality control (QC) sample preparation
The standard stock solutions of CRT analyte and propranolol internal standard were prepared at 1000 μg/mL in methanol. With that solution a working solution containing the adequate concentrations of all the analytes was prepared in methanol: water (50:50) to spike the aqueous humour samples covering the linearity range (1–1000 ng/mL). A 100 ng/mL solution containing the IS propranolol was also prepared in methanol and diluted with methanol: water (50:50).
Calibration standard was prepared by spiking a pool of drug free aqueous humour (50 μL for each concentration) with the working standard solution and it was diluted to desire concentration range from 1 to 1000 ng/mL. The final concentration for each analyte was prepared to be ∗∗1.010, 2.900, 22.136, 208.378, 420.378, 638.000, 850.000, and 1000.000 ng/mL. Calibration standards were processed according to the sample preparation procedure and analysed by the UPLC–MS/MS method. QC samples were prepared independently at three levels; ∗∗800.000 ng/mL (HQC, high quality control), 400.000 ng/mL (M2QC, middle quality control), 34.059 ng/mL (M1QC, middle quality control) and 2.900 ng/mL (LQC, low quality control) and 1.010 ng/mL (LLQC).
2.5 Sample treatment
A volume of 50 μL aliquot of all rabbit aqueous humour was pipetted into a 1.5 mL eppendorf tube and spiked with 50 μL of a 100 ng/mL IS solution propranolol. Protein precipitation was carried out using 200 μL formic acid (2% v/v) solutions. After vortex mixing, it was transferred to a 5 mL glass vial and 3 mL of ethyl acetate was added, shaken and centrifuged at 18,000 rpm for 10 min. The supernatant was transferred to a 5 mL glass vial and evaporated to dryness under nitrogen stream at 60 °C. The residue was reconstituted with 500 μl of mobile phase (acetonitrile: 2 mM ammonium acetate 90:10 v/v) and vortexed. The solution was transferred into the clean auto sampler vials and 10 μL was injected into UPLC/MS system for analysis. All the aqueous humour of rabbit samples were stored at −20 °C, allowed to thaw at room temperature and were vortexed for 5 min prior to sample preparation.
2.6 Method validation
The method was validated according to internationally accepted recommendations (US Food and Drug Administration, 2012). The method was validated in terms of linearity, precision, accuracy, extraction recovery, matrix effect and stability.
2.6.1 Specificity
The selectivity of the method was evaluated by comparing the chromatograms of six different batches of blank aqueous humour with the corresponding spiked plasma (LLQC samples) to ensure no interfering peak of analyte and I.S. from endogenous aqueous humour components.
2.6.2 Linearity and sensitivity
The calibration standards of concentrations 1.010, 2.900, 22.136, 208.378, 420.378, 638.000, 850.000, and 1000.000 ng/mL were used to assess linearity, which were prepared according to Section 2.4. Calibration curves were plotted as the peak area ratio (drug/internal standard) versus the analyte concentration. Least-squares linear regression method was used to determine the slope, intercept and correlation coefficient.
2.6.3 Accuracy and precision
The precision and accuracy of this analytical method were evaluated by assay QC samples at 5 levels (LOQC, LQC, M1QC, M2QC and HQC concentrations). The QC samples were analysed six times a day to evaluate intra-day precision and accuracy. The same procedure was performed once a day for three consecutive days to determine inter-day precision and accuracy. The accuracy and precision were calculated using the formula published elsewhere (Lakhani et al., 2005) and the criteria for acceptability of the data included accuracy within ±15% standard deviation (SD) from the nominal values and precision within ±15% relative standard deviation (RSD) (Bioanalytical Method Validation, US Department of Health and Human Services Food and Drug Administration, Center for Drug Evaluation and Research (CDER) 2001) (Guidance for Industry, 2001).
2.6.4 Extraction recovery and matrix effect
The extraction recovery of CRT through the liquid–liquid extraction method was evaluated at four QC concentrations: 2.900 (LQC), 34.059 (M1QC), 400.000 (M2QC) and 800.000 (HQC) ng/mL (n = 6). Extraction recovery was determined by comparing the peak areas obtained from blank aqueous humour spiked with analyte before the extraction with those from samples to which analyte was added after the extraction. Matrix effect was assayed to compare the peak areas of blank aqueous humour extracts spiked with analyte with those of the standard solutions dried and reconstituted with mobile phase.
2.6.5 Stability assessment
The stability study was performed to evaluate the stability of the analyte in rabbit aqueous humour under the following conditions: short-term stability at room temperature for 24 h; long-term stability at −80 °C for 30 days, freeze thaw (three cycle at 20–25 °C) cycles for three consecutive days and post preparative stability of the processed samples kept under the auto sampler conditions (10 °C) for 24 h. All stability testings in aqueous humour were performed at the LQC and HQC concentrations (n = 6). All samples were compared with regularly analysed samples with the same amount of analyte and percentage stability was determined by the following formula. Stability was determined by statistical comparison of the calculated concentrations (t-test, 95% significance level).
2.7 Forced degradation of CRT
A stock solution containing 50 mg CRT in 50 mL methanol was prepared. This solution was used for forced degradation.
2.7.1 Preparation of acid- and base-induced degradation product
To the volume of 20 mL of methanolic stock solution, 5 mL of 5 N HCl and 5 mL of 5 N NaOH were added separately. These mixtures were refluxed for 6 h at 90 °C. The forced degradation in acidic and basic media was performed in the dark in order to exclude the possible degradative effect of light. The resultant solution (400 ng/mL) was injected to UPLC–MS/MS system.
2.7.2 Preparation of hydrogen peroxide-induced degradation product
To the volume of 20 mL of methanolic stock solution, 5 mL of hydrogen peroxide (30.0%, v/v) was added. The solution was heated in boiling water bath for 6 h to remove completely the excess of hydrogen peroxide. The resultant solution (400 ng/mL) was injected to UPLC–MS/MS system.
2.7.3 Photochemical degradation product
The photochemical stability of the drug was also studied by exposing the stock solution to direct sunlight for 24 h. The resultant solutions were appropriately diluted and 400 ng/mL was injected to UPLC–MS/MS system.
2.8 Application of the developed method for pharmacokinetic studies in ocular delivery of CRT
2.8.1 Preparation of CRT nanoparticles and characterisations
Chitosan (CS) nanoparticles were prepared according to the procedure first reported by Calvo et al. based on the ionic gelation of CS with sodium tripolyphosphate (TPP) anions (Calvo et al., 1997b). Chitosan was dissolved in 1% acetic aqueous solution at various concentrations (0.05–0.30%). 4 mL TPP aqueous solution with various concentrations (0.05% to 0.30%) was added into 10 mL chitosan solution, under magnetic stirrer at 1000 rpm for 45 min and the formation of chitosan–TPP nanoparticles started spontaneously via the TPP initiated ionic crosslink and coacervation mechanism. The nanoparticles were formed at selected chitosan to TPP weight ratios of 2.5:1. CRT nanoparticles were collected by centrifugation (15,000 rpm, 30 min), washed 3 times with Milli-Q water, finally resuspended in 4 mL of Milli-Q water and dried in a lyophilizer (Heto Drywinner, Germany).
2.8.2 Characterisation of chitosan nanoparticles
Chitosan nanoparticles were characterised for particle size, particle size distribution zeta potential, polydispersity index (size distribution) and morphology (Zetasizer® 3000-HS Malvern Instruments, UK) was used for measurement of particle size, particle size distribution, zeta potential and polydispersity index which is based on the Photon Correlation Spectroscopy (PCS) techniques. Samples were diluted to appropriate concentrations with deionised water. Triplicate samples were analysed and the mean value of the three was adopted. Morphological characteristics of the nanoparticles were examined using a high resolution Transmission Electron Microscope (TEM) machine (Philips CM 10, Holland) at an accelerating voltage of 100 kV. Data acquisition was done on the AMT Image Capture Engine. Freshly made nanoparticles were diluted with deionised water with adjusted pH close to neutral. One-drop sample was syringe placed on a carbon coated film 300 mesh copper grid, allowing sitting for 10 min until air-dried. The sample was stained with 1% phosphotungstic acid solution for 1.5 min and excess phosphotungstic acid was removed with filter paper before viewing for TEM.
2.8.3 Ocular Pharmacokinetic Study
The ocular pharmacokinetic study was performed on two groups of New Zealand albino rabbits (n = 6) V and the study was conducted as per the protocol and guidelines approved by the institutional animal ethics committee, Jamia Hamdard (approval No. 771). For the topical instillation, weighed amount of lyophilized CRT laden nanoparticles was dispersed in isotonic buffer to get 1%w/v solution. CRT solution (1%, w/v) was also prepared in the same vehicle. To the first group, CRT solution (50 μL) was instilled topically whereas to the second group CRT-nanoparticles (50 μL) were instilled. Formulations were instilled in both the eyes of rabbits and approximately 50 μL of aqueous humour was collected before instillation of formulations and post-treatment at 0.5, 1, 2, 4, 6, 12 and 24 h from one rabbit (both eyes) at each time point. Aqueous humour samples were collected in eppendorf tubes, sealed and stored at −20 °C until UPLC–MS/MS analysis. The aqueous humour samples were prepared as mentioned above. Pharmacokinetic parameters were calculated by non-compartmental analysis, also called as model-independent analysis using WinNonLin version 4.0 (Pharsight Corp., Mountain View, CA). Peak aqueous humour concentration (Cmax) and time of its occurrence (tmax) were read directly from the individual aqueous humour concentration–time profiles. Area under the concentration–time curve (AUC 0−t) was calculated according to the linear trapezoidal method.
3 Results and discussion
3.1 Optimisation of the UPLC/MS/MS method
A novel UPLC–MS/MS method was developed by using the different compositions of mobile phase, which affected the separation and ionisation of CRT and I.S. In the present study various mobile phases such as methanol, isopropanol were tried, but they did not show the chromatographic separation. Therefore, the optimisation of mobile phase components was critical. Various mobile phase composition such as methanol: water, methanol: formic acid (0.1%), methanol: 1, 2, 5 mM ammonium formate, acetonitrile: water, acetonitrile: formic acid (0.1%) and acetonitrile: 1, 2, 5 mM ammonium formate were tried but peak did not separate properly. Finally the optimised chromatographic separation of CRT, was achieved by using acetonitrile: 2 mM ammonium acetate buffer (90: 10, v/v), with a flow rate of 0.2 mL/min. Baseline separation of both CRT and IS was obtained within runtime of 3 min and fragments of high intensities were selected for ion monitoring. The full MS scan spectra for CRT showed protonated precursor [M+H]+ ions at m/z 293.18 and their product ion mass spectra (MS/MS) at m/z 237.1 respectively neutral loss of isobuthene from the [M+H]+ of CRT, which was selected for sensitive quantification of the drug (Figs. 2 and Fig. 1C) while I.S. response was showed at m/z 260.09 was which corresponds to the respective protonated molecular ions [M+H]+ with most abundant product ion (MS/MS) at m/z 183.04 (Figs. 2B and D). The retention times were 1.29 min for CRT and 2.38 min for I.S respectively and total chromatographic runtime was 3.0 min (Fig. 3A and B). Chromatogram of blank plasma (extracted and reconstituted) is shown in Fig. 3C.![Full scan product ion mass spectra of the [M+H]+ at m/z 293.18 (CRT) in the (A), m/z 260.09 (Propranolol IS) (B), product ion at m/z 237.13 (CRT) (C) and product ion at m/z 183.04 (Propranolol IS) (D).](/content/184/2017/10/2_suppl/img/10.1016_j.arabjc.2013.11.034-fig2.png)
3.2 Validation of the analytical method
3.2.1 Specificity
The selectivity was evaluated by comparing the chromatograms of blank aqueous humour, and spiked aqueous humour samples (LLOQ, 1.010 ng/mL) in pharmacokinetic studies. Comparison of spiked aqueous humour samples and blank aqueous humour showed no significant peaks interfering with the quantification of the analyte and I.S. and the representative chromatogram of aqueous humour samples showed similar chromatographic behaviour to QCs.
3.2.2 Linearity and sensitivity
The calibration curve was linear with regression correlation coefficients (r2 = 0.999) over the concentration range of 1.010–1000 ng/mL. The equation of the calibration curves was: y = 0.041x + 0.238, r2 = 0.999 where x represents the aqueous humour concentration of CRT and y is the peak area ratio of CRT to I.S. The limit of quantification (LOQ) was found to be 0.75 ng/mL in aqueous humour (S/N ⩾ 10), which was sufficient to support pharmacokinetic studies of CRT. The accuracy of the diluted samples of CRT was 99.38 ± 1.96 % at 400 ng/mL and 99.12 ± 2.01% at 800 ng/mL. The precision determined at each concentration level was within 5% of the relative standard deviation (%R.S.D.). The LOD and LOQ were determined as 0.25 and 0.75 ng/mL respectively for CRT in aqueous humour. Dilution study found that the assay was reasonable to quantify CRT in samples that exceeded the upper limit by an appropriate dilution.
3.2.3 Precision and accuracy back calculated concentrations
The intra-day and inter-day precision and accuracy for the tested concentrations (LLQC, LQC, M1QC, M2QC and HQC) were within acceptable limits, (Table1). Intra-day precision (expressed as percent relative standard deviation, % RSD) ranged from 1.53% to 4.19% and the inter-day precision ranged from 1.05% to 4.73%. The precision values were found to be within the accepted limits, which indicated that the method was reproducible. Intra and inter-day accuracy ranged from 94.45 ± 3.70 to 99.38 ± 1.96 % and 96.04 ± 0.99 to 99.57 ± 1.88 %, respectively, and found to be within the accepted limits. This indicated that the method was accurate and reliable.
| Conc. (ng/ml) | Measured conc. (Mean ± SD) (ng/mL) | Intra-day Precision (% RSD) | Accuracy (Mean ± SD, %) | Measured Conc. (Mean ± SD) (ng/mL) | Inter day Precision (% RSD) | Accuracy (Mean ± SD, %) |
|---|---|---|---|---|---|---|
| 1.010 (LOQQC) | 0.96 ± 0.02 | 1.53 | 95.05 ± 1.98 | 0.97 ± .01 | 1.05 | 96.04 ± 0.99 |
| 2.900 (LQC) | 2.92 ± 0.10 | 3.82 | 100.68 ± 3.45 | 2.83 ± 0.12 | 4.24 | 97.59 ± 4.13 |
| 34.059 (M1QC) | 32.17 ± 1.26 | 4.19 | 94.45 ± 3.70 | 32.74 ± 1.55 | 4.73 | 96.13 ± 4.35 |
| 400.000 (M2QC) | 397.50 ± 7.82 | 1.97 | 99.38 ± 1.96 | 398.26 ± 7.53 | 1.98 | 99.57 ± 1.88 |
| 800.000 (HQC) | 795.49 ± 12.71 | 1.60 | 98.81 ± 2.34 | 792.94 ± 16.08 | 2.03 | 99.12 ± 2.01 |
RSD (%): relative standard deviation.
3.2.4 Extraction recovery and matrix effect
Extraction recovery and matrix effect were determined as described in Section 2.5.4 and the data are shown in Table 2. The extraction recovery of the compound from CRT matrix was 93.98 ± 3.34 to 95.90 ± 2.64 % at LQC, M1QC, M2QC and HQC concentrations. As for ionisation, the peak areas of CRT after spiking evaporated aqueous humour samples at four (LQC, M1QC, M2QC and HQC concentrations) concentration levels were comparable to neat standard solutions (ranged from 95.34 ± 7.86 % to 97.54 ± 3.57%), suggesting that there was no measurable matrix effect interfered with CRT determination in rabbit aqueous humour sample.
| Conc. (ng/mL) | Recovery (Mean ± SD) (%) | Matrix effect (Mean ± SD) (%) | % RSD |
|---|---|---|---|
| 2.900 (LQC) | 94.43 ± 6.67 | 95.34 ± 7.86 | 3.68 |
| 34.059 (M1QC) | 93.98 ± 3.34 | 96.12 ± 5.56 | 3.98 |
| 400.000 (M2QC | 95.90 ± 2.64 | 96.72 ± 4.78 | 2.67 |
| 800.000 (HQC) | 95.32 ± 3.98 | 97.54 ± 3.57 | 2.34 |
3.2.5 Stability study
The stability of spiked aqueous humour sample was done at different storage conditions such as short term, long-term, freeze–thaw, and post processing stability. The study was performed at two QC levels i.e. LQC and HQC. The results were found to be that CRT was stable during all storage condition and data shown in Table Table 3. The recovery of the analytes relative to that at time zero was reviewed. After short term for 24 h storage the recovery of CRT was 99.31% at LQC and 98.79% at HQC. After 1 month of storage (Long-term stability) the recovery of CRT was 97.95 % at LQC, and 97.87% HQC. After 1, 2 and 3 cycles of freeze–thaw (freeze–thaw stability), the average recovery of CRT was 98.63 % at LQC and 97.61% at HQC. The recovery (post processing stability) of CRT was 98.97% at LQC and 98.64% (HQC). The above results showed that a sensitive, reproducible and robust method for the analysis of CRT in aqueous humour had been developed and validated.
| Conc. (ng/ml) | Stability | Mean ± SD (ng/ml) | % Drug remaining | % RSD |
|---|---|---|---|---|
| LQC (2.900 ng/mL) Initial (0 h) | Initial (0 h) | 2.92 ± 0.02 | 100 | 0.68 |
| Short-term stability (25 °C, 24 h) | 2.90 ± 0.03 | 99.31 | 1.03 | |
| Long-term stability (−80 °C, 30 days) | 2.86 ± 0.02 | 97.95 | 0.70 | |
| Three cycles freeze–thaw stability (three cycles, −20 °C to 25 °C) | 2.88 ± 0.03 | 98.63 | 1.04 | |
| Post-preparative stability (10 °C, 24 h) | 2.89 ± 0.01 | 98.97 | 0.34 | |
| HQC (800.0 ng/mL) | Initial (0 h) | 795.49 ± 12.71 | 100 | 1.59 |
| Short-term stability (25 °C, 24 h) | 785.87 ± 8.54 | 98.79 | 1.09 | |
| Long-term stability (−80 °C, 30 days) | 778.54 ± 6.32 | 97.87 | 0.81 | |
| Three cycles freeze–thaw stability (three cycles, −20 °C to 25 °C) | 776.45 ± 10.26 | 97.61 | 1.32 | |
| Post-preparative stability (10 °C, 24 h) | 784.74 ± 6.43 | 98.64 | 0.82 |
3.3 Stress degradation
3.3.1 Studies under acidic and basic conditions
After 6 h exposure in 5 N HCl and 5 ml of 5 N NaOH at 90 °C, additional mass peak was observed, which revealed the degradation of the drug. The peak observed in mass chromatogram is m/z 149.002 for acid degradation (Fig. 4A) and m/z 128.9315, 231.9315, and 245.073 for base degradation shown in (Fig. 4B). These masses were not observed in standard mass chromatogram shown in Fig 2A. In acid stress study the parent peak m/z 293.1372, and daughter ion peak m/z 273.0615 are present but in base stress studies, these peaks were absent, it showed the CRT is highly sensitive to base as compared to acid.
3.3.2 Oxidative stress studies
After exposure in 30%v/v H2O2 in a water bath for 6 h, one additional mass peak at m/z 128.9315 was observed but its intensity was less as compared to parent and daughter ion peak (m/z, 293.1372, 237.0615) (Fig. 4C). This revealed that the drug possesses lower sensitivity to oxidative stress.
3.3.3 Photochemical stress study
The photochemical stability of the drug was also studied by exposing the stock solution to UV light for 24 h. There was no additional mass peak observed. The mass chromatogram was similar to standard mass chromatogram (Fig. 4D). Therefore the CRT is insensitive to UV light.
3.4 Preparation and characterisation of CRT nanoparticles
The preparation of CRT loaded chitosan (CS) nanoparticles was based on the ionic inter-action of a positively charged CS-CRT solution and negatively charged TPP solution (Calvo et al., 1997b). The charge density of both CS and TPP solution has a great effect on the ionic interaction. It is well known that the charge density is greatly influenced by the pH value of solution. The nanoparticles were characterised as particle size, zeta potential, PDI, assay of the drug and morphology by TEM. The particle size (Fig. 5A), zeta potential, PDI and entrapment efficiency of optimised formulation were 169.9 ± 3.32 nm, 34.23 ± 1.21 mV, 0.31 ± 0.04 and 70.34 ± 1.54% respectively. The morphology of nanoparticles was spherical in shape (Fig. 5B).
3.5 Pharmacokinetic study
The validated UPLC–MS/MS analytical method was successfully used to quantify CRT from drug solution and CRT-nanoparticles in aqueous humour of rabbit. The high sensitivity of the present method allowed to measure aqueous humour concentrations up to 24 h post dosing. The formulations (CRT – solution and CRT – nanoparticles, dose: 1% w/v; 50 μL) were instilled topically to New Zealand rabbit’s eye and the developed UPLC–MS/MS method was applied to determine their in vivo pharmacokinetics at predetermined time intervals. Fig. 5 shows the aqueous humour concentration – time profiles of carteolol after instillation. The pharmacokinetic parameters in aqueous humour such as AUC0−t, AUC0−∞, Cmax, Tmax, and t1/2 obtained from the pooled concentration- time, data are shown in Table 3. The group was treated with the solution, the drug was undetectable in aqueous humour after 6 h, due to rapid precorneal drainage of solutions. In contrast, in the group treated with CRT – nanoparticles the drug was detected in aqueous humour for at least 24 h (27.21 ± 3.35 ng/mL). The increased t1/2 value of CRT – nanoparticles (4.72 h) indicated its sustained release ability. The AUC0−t applied by CRT – nanoparticle formulation (AUC0−t: 5265.18 ± 25.76 ng/mL. h) was 5.61 times higher, compared with CRT-solution (AUC0−t 939.24 ± 19.23 ng/mL). It could also be seen that maximum level (Cmax) of carteolol in aqueous humour of CRT –nanoparticles was approximately 2.1-fold (581.76 ± 7.52 ng/mL) higher than CRT-Solution (312.54 ± 5.34 ng/mL). It is due to the positively charged mucoadhesive nanoparticles which interact with negatively charged cornea and prolonged ocular contact time, thus maintaining prolonged corneal drug concentration gradient because of chitosan having bioadhesive property. The results also indicated that the chitosan nanoparticles are able to deliver optimum levels of drug into aqueous humour and show promise for ocular delivery for management of glaucoma. (See Fig. 6 and Table 4).
| Pharmacokinetic parameter | CRT-solution | CRT-nanoparticles |
|---|---|---|
| Cmax (ng/mL) | 312.54 ± 5.34 | 581.76 ± 7.52 |
| Tmax (h) | 1.00 ± 0.12 | 2.00 ± 0.16 |
| T1/2 (h) | 2.52 ± 0.23 | 4.72 ± 0.30 |
| AUC0−t (ng/mL. h) | 939.24 ± 19.23 | 5265.18 ± 25.76 |
| AUC0−∞ (ng/mL. h) | 964.46 ± 24.83 | 5517.78 ± 30.56 |
4 Conclusion
This paper describes the stability indicating method development, validation and application of a UPLC–MS/MS method for the quantitative analysis of CRT from CRT-solution and CRT – nanoparticle in rabbit aqueous humour. The extraction of CRT was done by a fast liquid–liquid extraction method. The method was proven to be highly sensitive, accurate and selective, making it suitable for high-throughput quantitative analysis of CRT with short run time. The drug was found to be stable in acid, oxidative and photolytic (UV-light) conditions and well degraded in basic medium. The pharmacokinetic parameters of nanoparticles in aqueous humour were found to be AUC0−t, (5265.18 ± 25.76 ng/mL. h) Cmax, (581.76 ± 7.52 ng/mL), and t1/2, (4.72 h) when compared to the drug solution. The high concentration of drug was retained in aqueous humour for a longer period of time due to the increased corneal retention when compared to drug solution. So it has been successfully applied in pre-clinical ocular pharmacokinetic of novel ophthalmic formulations.
Acknowledgments
The authors are thankful to Indian Council of Medical Research (India), for providing Senior Research Fellowship (Iris ID- 2011- 03390). They are also grateful to Otsuka Pharmaceuticals Ltd (Japan) for providing a gift sample of pure drug.
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