Development and validation of simultaneous quantification method for gemcitabine and betulinic acid: augmenting industrial application

Abstract

Combinatorial treatment utilizing a nucleoside analogue gemcitabine (GEM), with a characteristic pentacyclic triterpenoid betulinic acid (BET), has exhibited empowering adequacy in the therapy of cancer. It lessens the advancement of collagen and upgrades the saturation of tumour medicines. With the advancement in nanotechnology, the co-loaded formulation urges for a validated method of estimation. The purposed work entails a robust, simple, and economical analytical method for the simultaneous estimation of GEM and BET through RP-HPLC. Orthophosphoric acid (0.1%)—acetonitrile was considered as the mobile phase for the detection of GEM and BET at 248 nm and 210 nm with retention times of 5 min and 13 min, respectively. The method was further validated as per the regulatory guidelines with all the parameters found within the limit. The developed method with adequate resolution and quantification was found to be linear, accurate, precise, robust, and stable with an intra- and inter-day variability of less than 2%. The method was found specific for GEM and BET with no matrix interference of drug-spiked FBS samples. To demonstrate the applicability of the developed method, a nano-formulation containing GEM and BET was prepared and assessed for various parameters including encapsulation efficiency, loading efficiency, drug release, and drug stability. The method developed can be a possible tool for the simultaneous quantification of GEM–BET in analytical and biological samples.

Introduction

The current treatment of cancer with a single drug requires a considerable amount of drug to be administered which often experiences challenges such as low bioavailability, decreased absorption, resistance, and toxicity (Nagai and Kim 2017). The targeting of more than one site in a cancerous cell can be an alternative to the current chemotherapy (Liang et al. 2010). Further, the peculiar heterogeneity of tumor cells with the adaptive mechanism for drug resistance recommends the use of combined therapy for cancer (Lim and Ma 2019). The diversified mechanism of proliferation and metastasis of the tumor cell, such as angiogenesis, DNA replication, cancer cell survival pathways, and tumor stromal-epithelial interactions, calls for effective management using different natural as well as synthetic chemotherapeutic drugs alone, or in combination to target different sites at a time (Chatterjee and Bivona 2019). Thus, various pharmacologically active secondary metabolites of natural origin such as betulinic acid, and kinase inhibitors such as imatinib and lapatinib etcetera are presently being used to achieve synergism in a clinical setting (Lu et al. 2020).

In combination therapy, the drugs acting on different target sites often possess different chemical properties which affect their pharmacokinetic and pharmacodynamics attributes even when co-loaded in the nano-formulation (Chanda et al. 2014). Though, GEM and BET have different chemical properties, yet reported potential synergism for the treatment of cancer (Saneja et al. 2019). GEM being hydrophilic have an easy bioavailability, but the poor solubility of BET restricts its delivery (Momin et al. 2017; Khan et al. 2018). Several works of literature are available for the individual estimation of GEM and BET but not in combination. In comparison to GEM, the quantification of BET is complex in terms of extraction, sample preparation, and processing from different formulations (BET is reported to depict low sensitivity) and biological samples thus embarks a major challenge in the development of the analytical method. These differences in the chemistry of drugs warrant an obstacle for the exact quantification in different formulations. Further, for the developed dosage form, the quantification of the drug content is necessary to meet the regulatory guidelines (Falkowski et al. 1987). The sensitivity of the method and instrumental configuration used by Sahu et al. (2018), is comparable to the present study, but the applicability of the method with biopharmaceutical prospects was not discussed (Sahu et al. 2018). Patel et al. (2016), provided a pH-dependent analysis of gemcitabine but lacks a combination (Patel et al. 2016). The method developed by Nuland et al. (2018), on LC–MS was more sensitive but adds an economic burden (Nuland et al. 2018). For betulinic acid, the most cited method developed by Zhao et al. (2007), have a LOD limit of 6.2 µg/ml rendering it less applicable in terms of biological and bio pharmacokinetic applications (Zhao et al. 2007). A similar method was developed by chanda et al. (2014), for the quantification of betulinic acid with a LOD limit of approximately 0.9 µg/ml but lacking pharmaceutical applicability in biological samples (Chanda et al. 2014). The previously reported methods compromise either sensitivity or economic pitfalls or are intended for the estimation of the individual drug but not for combination (Fig. 1).

Fig. 1.

Chemical structure of A Gemcitabine B Betulinic acid

Given the said consequences, we envisaged developing an RP-HPLC method for the simultaneous analysis of the GEM and BET. The method was initially validated in aqueous solutions according to the requirements of the ICH in terms of linearity, measurement range, precision, accuracy, and stability of drugs over time. The specificity was established in a mixture of dissociated nanoformulation in water, as well as in fetal bovine serum (FBS). The method developed was further subjected to demonstrate its applicability and reliability for the quantification of both the drugs, such as assay, encapsulation efficiency, stability assessment, dissolution, pharmacokinetics, forced degradation, and detoxification.

Materials and methods

Materials

Gemcitabine was a gift for research purposes from Sun Pharmaceutical Industries Limited, Gurugram, Haryana, India, and was utilized as obtained. Betulinic acid was purchased from Sigma-Aldrich. HPLC grade of acetonitrile, methanol, ortho-phosphoric acid, and different solvents used, were procured from Merck Pvt Limited, Mumbai, India. Triple distilled water was obtained from in house Milli-Q unit (Millipore, Bedford, MA, USA).

Method

RP-HPLC instrumentation and chromatographic conditions

HPLC instrument (Shimadzu) furnished with LC 10 ATVP isocratic pump with Rheodyne model 7125 injector (Cotati, CA, USA) containing 20 µL circle and SPD-M10 AVP UV-PDA detector (Shimadzu) was utilized. 20 µL of sample volumes were injected into the RP-18e Lichrospher®100 (250 mm × 4.6 mm, 5 μm) column. Chromatographic partition was conducted in an isocratic scheme. The mobile phase was comprised of 0.1% orthophosphoric acid (OPA) buffer and Acetonitrile (ACN) mixed in a proportion of 20:80 and a flow rate of 1 mL/min. All the solvents were filtered and sonicated by utilizing a bath sonicator at room temperature for 10 min before the experiment. The eluents were observed at 248 nm and 210 nm for GEM and BET, respectively.

Preparation of standard and sample solutions

The appropriate amount of GEM and BET was solubilized in methanol to make a stock of 1 mg/mL from which the secondary stock solution of 500 μg/mL was prepared. The stock was kept at – 20 °C till further use. The working solution of 5 µg/ml for GEM and BET from the secondary stock solution was prepared.

Validation methodology

The assay method for GEM and BET was validated as per ICH guidelines for different validation parameters as mentioned below (Guideline 1996; US FDA 2000).

Linearity

To assess the linearity of the method developed, the stock solution was diluted to a concentration of 500 µg/mL, which was sequentially diluted to prepare six sample solutions containing equivalent concentrations of GEM and BET ranging from 0.1 to 500 µg/mL. All GEM and BET constituting sample dilutions were injected into HPLC separately. The obtained peak area and respective concentrations were plotted. The curve so acquired was then fitted utilizing linear regression and the r² value was determined (Mangamma et al. 2012).

Accuracy and precision

For examining the accuracy of the proposed method, three different levels of the test samples, including 50% (QC low), 100% (QC mid), and 150% (QC high) for GEM and BET were injected and analysed in triplicate. Results were processed and analysed against percent recovery and percent relative standard deviation (%RSD). Furthermore, for estimation of the precision of the developed method six distinct samples of equal concentration (5 µg/mL) were prepared and analysed. The peak area for both GEM and BET acquired were processed for the estimation of percent recovery and percent RSD with reference to ICH guidelines. (Singh et al. 2015).

Limit of detection (LOD) and limit of quantification (LOQ)

LOD is defined as the lowest limit of the strength of the analyte that can be detected, whereas LOQ is the least concentration of analyte that can be resolved quantitatively with accuracy and precision. Both parameters were computed utilizing the standard deviation of the response (five concentrations from the linearity range, from 0.1 to 5 µg/ml for DOX and 0.5–50 µg/ml for BET) and slope obtained from the calibration curve for both GEM and BET, fitting in the standardized formula as reported in the literature, mentioned below (Singh et al. 2017)

$$\text{LOQ}=\left(10\ast \alpha \right)/m$$

$$\text{LOD}=\left(3.3\ast \alpha \right)/m$$

where α is the standard deviation of blank injection and m represents the slope of the calibration curve.

Standard solutions stability

The stability was evaluated to assess changes in GEM and BET concentration as an element of time and temperature. The stability studies (long-term and short-term) were conducted by preparing a test sample of 5 µg/mL kept at refrigerated temperature (4–8 ºC) and room temperature (25 ºC) for up to 3 days. The drug concentration in the samples and % recoveries were calculated. The stability was inferred by comparing the initial concentrations obtained at day 0 (Srivastava and Chaturvedi 2010).

Method robustness

The robustness of the proposed method was assessed by analysing a 5 µg/mL test sample, by intentionally altering the flow rate (from 1 to 0.95 and 1.05 mL/min), the composition of the mobile phase, and the column temperature (from 25 to 20 and 30 °C) to a variation of ± 5%. The impact of the altered robustness parameters on area, retention time etcetera, was acknowledged by the different peaks observed at varied parameters and was reported as percent recoveries (Taralkar and Chattopadhyay 2012).

Preparation of nanoformulation

GEM and BET-loaded nanoformulation were fabricated utilizing a solvent evaporation strategy. The drugs (GEM and BET) were dispersed in ethanol by mixing followed by sonication to form a clear ethanolic phase. Subsequently, the ethanolic phase was added dropwise to an aqueous phase containing 2% tween 80 and processed at low temperature utilizing a T 25 digital ultra-turrax homogenizer (IKA, Germany). The mixture was kept stirring till evaporation of the non-aqueous solvent. The volume of the obtained formulation was made up to 10 mL with triple distilled water. Placebo nano-formulation was also prepared without GEM and BET, following a similar protocol as mentioned previously. The nanoformulation was further characterized for various pharmaceutical attributes such as size, polydispersity index (PDI), and zeta potential utilizing dynamic light scattering (Kim et al. 2019; Chivere et al. 2020).

Specificity of analysis in presence of FBS

The specificity of the method was determined in blank FBS samples and FBS samples spiked with known concentrations of GEM and BET (250 μg/mL of GEM plus 500 μg/mL of BET). 900 µL FBS was spiked with 100 µL of the above-mentioned concentration of GEM and BET. The sample was incubated for 2 h, followed by the addition of acetonitrile (intended for protein precipitation) and methanol (for solubilizing both drugs), and vortexed for 30 min. The supernatant was collected, processed, and analysed using a developed method through HPLC. All samples were analysed in triplicate for any interference in the peaks at respective retention times for GEM and BET against blank FBS and formulation components (Lanz et al. 2007).

Statistical analysis

All the experiments were performed in triplicate. Statistical analysis of the obtained results was performed utilizing Graph Pad Prism software, 5.0 (Graph Pad Software Inc., CA, USA) (Savadkouhi et al. 2017).

Results

The GEM and BET both have contrasting physical properties; GEM is hydrophilic and BET is hydrophobic. Initially, the method development process involved segregation and selection of chromatographic conditions with adequate resolution and precise analysis from the published literature and reported chemical characteristics of the drugs. In accordance with the available information and the trials conducted, the RP 18e Lichrospher®100 (250 mm × 4.6 mm, 5 μm particle size) column exhibited fair separation. A final mobile phase composed of 0.1% w/v OPA aqueous solution and ACN in the ratio of 20:80 resulted in good resolution of GEM and BET at 5.3 min and 12.8 min respectively. Further optimization of the developed method validates its reproducibility for simultaneous estimation of GEM and BET under specified condition (Fig. 2).

Fig. 2.

Representative chromatograms obtained by employing the developed RP-HPLC method for the resolution of GEM and BET: A GEM at 248 nm (RT 5.37 min, 10 µg/mL); and B BET at 210 nm (RT 12.53 min, 5 µg/mL)

Method validation

Linearity

The linearity samples of GEM and BET ranging from 0.1 to 500 µg/mL were injected into the HPLC system. The peaks for both GEM and BET at the 5.3 and 12.8 retention times were observed. The peak areas obtained were graphically analysed as a function of analyte concentration followed by least-squares linear regression analysis. The correlation coefficient (r²) for both drugs was observed to be 0.9999 (Fig. 3).

Fig. 3.

Linearity obtained by injecting analytical samples constituting GEM and BET in equivalent concentration within the range of 0.1–500 µg/mL of GEM and BET expressed with equations and coefficient of correlation

Accuracy and precision

The accuracy and precision were measured for the samples constituting both GEM and BET. The samples for accuracy testing were quantified and processed to obtain the % recovery. The method precision was evaluated by analysing samples at a concentration of 5 μg/mL for both drugs. The results for accuracy and precision were expressed as % RSD (Table 1). The variations were measured in terms of % RSD and found to be less than 1% (within the resolution limit) for both analyte. Accuracy results (Table 1) showed that the method was accurate with % recovery in the range of 99–102% for both drugs. The precision results are accurate as presented in Table 1.

Table 1.

Accuracy and method precision expressed as percent recovery and percent relative standard deviation for GEM and BET

Accuracy (n = 3)
Concentration [µg/mL]	Mean area		Mean amount recovered (µg)		% Recovery		Standard deviation		% Relative standard deviation
	GEM	BET	GEM	BET	GEM	BET	GEM	BET	GEM	BET
5	78,657	29,787	2.57	2.52	101.74	99.86	0.02	0.01	0.86	0.48
10.0	151,613	55,563	4.95	4.71	99.09	99.02	0.03	0.06	0.64	1.39
15	304,537	118,318	9.95	10.02	99.29	99.98	0.05	0.01	0.48	0.06

Method precision
Sample	Concentration [µg/ml]	Weight taken (mg)		Area		Mean amount recovered (mg)		% Recovery
		GEM	BET	GEM	BET	GEM	BET	GEM	BET
1	5	5.13	4.86	1,57,886	55,749	5.16	4.76	100.58	97.16
2	5	4.99	5.03	1,52,524	57,917	4.98	4.95	99.89	97.52
3	5	5.01	4.93	1,52,983	56,897	5.00	4.86	99.79	97.75
4	5	4.92	4.9	1,51,705	55,900	4.96	4.78	100.77	96.62
5	5	5	4.98	1,53,514	56,645	5.02	4.84	100.34	96.34
6	5	4.9	4.85	1,50,773	55,464	4.93	4.74	100.55	96.86
Average	5	5	5	1,53,231	56,429	5	5	100	97
Standard deviation				2477.13	911.40	0.08	0.08	0.40	0.54
% Relative standard deviation				1.6	1.6	1.6	1.6	0.4	0.6

Limit of detection (LOD) and limit of quantification (LOQ)

LOD and LOQ, for both drugs, were determined following the method described previously. The standard deviation and slope values were obtained by plotting a graph between area and concentration. These values for slope and standard deviation are applied in the equation to calculate LOD and LOQ. It was found that the LOD was 0.417 and 1.183 μg/mL, while LOQ was found 1.264 and 3.571 μg/mL for GEM and BET, respectively.

Standard solution stability

The stability of the solution was analysed by storing the methanolic solutions of the GEM (5 μg/mL) and BET (5 μg/mL) at 5 °C and 25 °C. The stability was assessed both intra-day and inter-day up to 3 days. As shown in Table 2. All solutions were found to be stable and negligible degradation was observed.

Table 2.

Stability expressed in % recovery and % relative standard deviation for GEM and BET after storage at 5 °C and 25 °C up to 3 days

Intraday (5 °C)
Analyte	Time (hrs)	Concentration [µg/mL]	% Recovery	Standard deviation	%  Relative standard deviation
GEM	0	5	100.78	0.06	1.07
	12	5	100.42	0.05	0.82
BET	0	5	100.72	0.03	0.60
	12	5	101.98	0.08	1.48

Interday (5 °C)
Analyte	Day	Concentration [µg/mL]	% Recovery	Standard deviation	%  Relative standard deviation
GEM	1	5	100.78	0.06	1.07
	2	5	101.25	0.08	1.40
	3	5	100.89	0.07	1.15
BET	1	5	100.72	0.03	0.60
	2	5	100.72	0.03	0.60
	3	5	100.83	0.04	0.67

Intraday (25 °C)
Analyte	Time (hrs)	Concentration [µg/mL]	% Recovery	Standard deviation	%  Relative standard deviation
GEM	0	5	100.784	0.062	1.073
	12	5	100.992	0.030	0.060
BET	0	5	100.724	0.033	0.597
	12	5	101.286	0.048	0.866

Interday (25 °C)
Analyte	Day	Concentration [µg/mL]	% Recovery	Standard deviation	%  Relative standard deviation
GEM	1	5	100.78	0.06	1.07
	2	5	100.46	0.02	0.37
	3	5	100.95	0.00	0.03
BET	1	5	100.72	0.03	0.60
	2	5	100.68	0.02	0.44
	3	5	101.01	0.04	0.67

Method robustness

The robustness was assessed by deliberately varying experimental parameters. Standard solutions of 5 μg/mL of GEM and BET were prepared and analysed with deliberated changes in chromatographic parameters like flow rate, the composition of mobile phase, and column temperature. It was found that the recoveries of both drugs were within 99–102%. The results obtained were within the limit of acceptance (Table 3). However, irrespective of negligible change in percent recoveries, the retention time of the GEM and BET was deviated due to the change in flow rate and mobile phase composition.

Table 3.

Robustness expressed in % mean recovery and % relative standard deviation for GEM and BET

Parameter	Mean of area	% Mean recovery	Standard deviation	%RSD
GEM
Mobile phase (75:25)	1,47,425	99.61	0.015	0.252
Mobile phase (80:20)	1,48,821	100.55	0.053	0.910
Mobile phase (85:15)	1,47,800	99.86	0.025	0.427
Flow rate (0.9 ml/min)	1,49,147	100.77	0.062	1.064
Flow rate (1.0 ml/min)	1,50,343	100.99	0.071	1.219
Flow rate (1.1 ml/min)	1,47,460	99.64	0.016	0.268
Column Temp. (20 °C)	1,49,229	100.83	0.064	1.103
Column Temp. (25 °C)	1,49,715	100.59	0.055	0.942
Column Temp. (30 °C)	1,50,691	101.82	0.104	1.796
BET
Mobile phase (75:25)	61,101	101.35	0.058	1.040
Mobile phase (80:20)	60,583	100.48	0.023	0.421
Mobile phase (85:15)	60,767	100.79	0.036	0.644
Flow rate (0.9 ml/min)	61,266	99.51	0.014	0.259
Flow rate (1.0 ml/min)	62,144	100.93	0.041	0.745
Flow rate (1.1 ml/min)	61,599	100.05	0.007	0.121
Column Temp. (20 °C)	61,180	101.48	0.063	1.133
Column Temp. (25 °C)	63,467	100.95	0.042	0.757
Column Temp. (30 °C)	60,699	100.68	0.031	0.564

Specificity

The specificity of the method was evaluated by spiking the known concentrations of both drugs (250 μg/mL of GEM + 500 μg/mL of BET) in FBS. The result confirmed that no interference (as shown in Fig. 4) was observed at respective retention times for GEM and BET in the blank FBS. This resembles that the method is specific.

Fig. 4.

A Chromatogram of placebo (FBS) and B chromatogram of FBS spiked with GEM and BET. Chromatogram showing the specificity of the method, no interference was observed at 5.3 min and 11.9 min i.e. RT of GEM and BET

Preparation of nanoformulation

The GEM and BET nano-emulsified system with Tween 80 and Phosphatidylcholine was prepared and used to demonstrate the applicability of the developed method. The developed nanoemulsion was found to depict 155.13 ± 5.26 nm, 0.412 ± 0.08, and − 31.2 ± 0.46 mV as hydrodynamic diameter, Poly dispersity index and zeta potential respectively, ensuring the monodisperse system (Fig. 5).

Fig. 5.

Graph depicting the results of dynamic light scattering results obtained for three different diluted samples of developed nanoemulsified system: A Particle size distribution and B Zeta-potential distribution

Applicability of the method

Assay/analysis of GEM and BET in nanoformulation

To illustrate the applicability of the method in quantifying GEM and BET, a nanoformulation containing both drugs was prepared and the amount of these drugs in the formulation was estimated. The % assay of GEM and BET was revealed as 100.08 ± 0.03% and 99.03 ± 0.023%, of the label claim (theoretical drug concentration) which was deemed highly acceptable. The chromatogram depicted the selectivity of the method for quantification of GEM and BET in the developed formulation explaining the method’s utility (Fig. 6).

Fig. 6.

Assay chromatogram of GEM and BET (RT at 5.3 and 11.99 min, respectively). The formulation constitutes the combination of different excipients along with the API, resulting in different extraction process and therefore slight matrix effect in the method was observed

Drug encapsulation efficiency and drug loading

The main idea for the combination delivery approach is to deliver the amalgamation together to the target neoplastic cells. Aiming the same, for the GEM and BET, we developed a nanoformulation, comprising an average particle size of 158.37 ± 6.29 nm. Optimization of the prepared formulation requires precise analytical characterization of an encapsulated drug for the estimation of its physio-pharmaceutical properties including drug encapsulation efficiency (DEE) and drug loading (DL). Thus, the characterization was performed by the developed and validated RP-HPLC method. In the developed formulation, DEE was observed to be 93 ± 11.4% and 82.3 ± 6.1% for GEM and BET, respectively. The amount of drug loaded in the formulation was found to be 9.3 and 8.2 mg/mL, respectively.

Dissolution/drug release study

To investigate the release profile of the drugs co-loaded in the developed formulation, a dissolution study was performed utilizing the dialysis bag technique. 1 mL nanoformulation containing 10 mg/mL of GEM and BET was prepared and filled in the dialysis bag (12–14 kDa). The prepared dialysis bag was immersed in a medium containing 0.9% ringer solution (900 mL) and stirred at 50 ± 5 rpm maintaining temperature of 37.5 ± 0.5 °C. The samples were withdrawn at predefined time points and replenished to maintain sink condition and analysed using a developed method through HPLC and a composite release profile was plotted (Fig. 7).

Fig. 7.

Graph depicting drug release profile shown by GEM and BET containing nanoformulation in terms of percent cumulative drug release versus time. The nanoformulation developed has been subjected to release study with phosphate buffer saline at pH 7.4 as dissolution medium mentained at 37 °C

In vitro release kinetic modelling

The release profile of the formulation was analysed and statistically processed according to different kinetic models (Fig. 8). The dissolution profile represents the delayed release behaviour of the formulation. The kinetic models providing an understanding of the drug release mechanism from nanoformulation were applied to the obtained data and the goodness of the models was evaluated. It was found that the drug release profile initially resembled zero order followed by the first order, indicating Higuchi's kinetics, suggesting that the release is controlled through the diffusion of the drug from the core.

Fig. 8.

Graph demonstrating different kinetic models; A First order B Higuchi C Hixson-Crowell D Korsmeyer-Peppas. The kinetic modelling, equations and trend lines have been generated using Microsoft Excel

Short-term stability studies

As per guidelines, a drug substance ought to be evaluated under storage conditions that demonstrate its stability. The storage conditions and study period picked are adequate to cover the storage, transportation, and ultimate use of the formulation. For the same, the GEM and BET formulation was subjected to short-term stability studies for up to 30 days with percent assay assessment at 0, 7, 15, and 30 days. The recovery obtained during the stability assessment depicted deviation within limits as the % RSD of the recovery after 30 days was found to be 0.392%, 0.941% & 1.043%, and 1.439% for 5 and 10 µg/mL of GEM and BET, respectively (Table 4).

Table 4.

Short-term stability expressed in percent mean recovery and percent relative standard deviation for GEM and BET

Days	Formulation concentration (mg/mL)	GEM			BET
		Mean (%)	SD	%RSD	Mean (%)	SD	%RSD
0	5	100.485	0.034	0.486	100.241	0.048	0.923
	10	99.946	0.028	0.327	99.378	0.013	0.188
7	5	100.587	0.032	0.419	99.685	0.026	0.304
	10	100.254	0.052	0.983	100.403	0.024	0.259
15	5	99.442	0.022	0.217	99.821	0.391	0.745
	10	101.221	0.011	0.127	98.927	0.017	0.191
30	5	99.218	0.031	0.392	98.373	0.053	1.043
	10	98.487	0.049	0.941	98.248	0.068	1.439
Average		99.955	0.032	0.487	99.385	0.08	0.637

Forced degradation & detoxification study

GEM and BET were subjected to forced degradation studies to determine the applicability of the method in terms of resolution and drug content in samples subjected to a variety of stress-related conditions for up to 48 h. The GEM and the BET were subjected to acid (0.1N HCl), alkaline (1N NaOH), oxidation (3% H2O2), and heat (80 °C). The drug concentration obtained under varied stress conditions has been plotted in Fig. 9. By comparing the bars, we established that GEM and BET are sensitive to acid hydrolysis and heat. Hydrolysis under alkaline conditions is relatively slower.

Fig. 9.

Depiction of forced degradation studies of GEM and BET for a time period of 48 h. Both GEM and BET in triplicate were used for forced degradation studies. The gemcitabine was found to be more resistant towards the stress conditions as compared to betulinic acid. Both the drugs were almost susceptible and got decomposed on the addition of NaOH

The area under the peak of the GEM and the BET continued to decrease with time implying a time-dependent degradation profile with a different rate for each stress condition. For detoxification, the apparatus and glassware involved in the formulation of the nanoformulation encapsulating GEM and BET were cleaned. The cleaning solution was put separately in a beaker where it was detoxified using perchloric acid. The detoxified samples were collected and processed for quantitation of the GEM and BET. After 48 h the content of GEM and BET in samples were found below LOD.

Discussion

Cancer is one of the deadliest enemies of humans, often requiring combination therapy for effective management (Sharma et al. 2010). Both the heterogeneity of tumor cells and the acquired drug resistance contribute to the general inability of anticancer monotherapies to successfully eradicate it (Longacre et al. 2014). According to previously published studies, greater efficacy in the treatment could be obtained by delivering a combination of two chemotherapeutics or in combination with natural anticancerous analogues to the tumor tissue (Patra et al. 2018). The development and optimization of the formulation require precise analytical characterization in terms of its physicochemical properties including entrapment and loading, which can be estimated using the validated reverse phase HPLC method. There are previously reported methodologies to quantify both GEM and BET individually but not simultaneously. Even the reported methods which involved the estimation of individual drugs are also highly sophisticated and expensive. Further, the sensitivity of BET requires sophisticated analytical instrument and tedious treatment processing which pose obstacle in its detection, both individually and simultaneously. The unstability of GEM and BET in aqueous media prompted us to prepare the sample solutions in methanol and both drugs were evaluated by UV spectrophotometer for exact absorption maxima, which were found at 210 nm and 248 nm, respectively. This was optimized and used in RP-HPLC as the detection wavelength. For mobile phase selection, various compositions of mobile phases both polar and non-polar solvents including methanol, acetonitrile, water, buffers of glacial acetic acid, and phosphoric acid with and without ion pairing agents were tried. Apart from this, different chromatographic parameters including, mobile phase ratio, column type, and column temperature, at varied flow rates and gradient systems were evaluated. The optimized mobile phase i.e., 0.1% OPA and acetonitrile in a ratio of 20:80 was selected based on improved detection, peak resolution, and peak shape of both drugs. The choice of mobile phase was optimized using different experiments and available chemical properties of both the drugs reported in the literature. In accordance with the chemical affinity and PKa data available, the polar solvent was ascertained to be acidified water that prevents the ionization of both GEM and BET resulting in the required affinity for the stationary phase. Further, the extent of acidification was also varied to find the best possible combination to deliver the intended chromatographic conditions. With an alteration concentration of phosphoric acid, the shape and sensitivity of the drug was reduced which can be reasoned by the ionization concept. The method was validated under the ICH guidelines. The column temperature was not found to be a critical parameter for this analysis and hence to avoid any noise variation or signal shifting, it was set at 25 °C as the standard temperature.

Conclusion

A novel and validated RP-HPLC method for the simultaneous analysis of combination regimens bearing GEM and BET has been developed. The method was validated according to ICH guidelines with appropriate resolution and quantification. The method proved to be linear, accurate, precise, robust, and stable with an intra- and inter-day variability of less than 2%. The method was found specific for both analyte without interference in blank as well as its spiked FBS samples. The method was further demonstrated for various industrial applications including dissolution, detoxification, and forced degradation.

Author contributions

AKT did method development and validation, a few applicability studies and wrote a manuscript. PKY, RS and RR performed proof of applicability by stability assessment, dissolution, forced degradation and detoxification studies. MNA performed the nanoformulation development assay and encapsulation efficiency study. MKC conceptualized and supervised the study, analysed the data and edited the manuscript.

Data availability

The data files are available from the corresponding author upon reasonable request.

Declarations

Conflict of interest

Regarding the order of authorship or with any external agency, all authors declare no conflict of interest. We also declare that the study does not represent any data from human or animal source.

Declaration of research involving human participants and/or animals

We declare that the study does not represent any data from human or animal source.

Informed consent

NA.