1 Introduction

Lidocaine HCl (LID) and Diltiazem HCl (DIL) are two distinct pharmaceutical compounds with different chemical compositions and therapeutic actions, yet both are crucial in medical treatment of anal fissure. LID, chemically identified as 2-(diethylamino)-N-(2,6-dimethylphenyl) acetamide hydrochloride with the empirical formula C14H23ClN2O, functions primarily as a local anaesthetic and cardiac depressant used to manage arrhythmias [1]. On the other hand, DIL, chemically [(2S,3S)]-5-[3-(dimethylamino)ethyl]-2-(4-methoxyphenyl)-4-oxo-2,3-dihydro-1,5-benzothiazepine-3-yl]acetate hydrochloride with the empirical formula C22H27ClN2O4S, acts as a calcium-channel blocker and vasodilator, relaxing smooth muscles in blood vessels [2]. Structures of LID and DIL have been shown in Fig. 1a and b, respectively.

Fig. 1
figure 1

a Chemical structure of Lidocaine HCl. b Chemical structure of Diltiazem HCl

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The combination of LID and DIL in rectal formulations is particularly beneficial for treating anal fissures [3], offering both therapeutic relief and symptomatic improvement. LID provides numbing effects to alleviate pain, while DIL works to relax smooth muscles, aiding in healing and reducing discomfort.

Analytical techniques for estimating LID and DIL individually have been extensively documented in the literature [4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21], including methods such as ultraviolet spectroscopy, high performance liquid chromatography (HPLC), reversed-phase HPLC (RP-HPLC), HPLC–MS/MS, and others. LID and DIL, both have been estimated in combination with other drugs by UV, HPLC and HPTLC, but only one UV method has been published for estimation of both simultaneously [22]. However, simultaneous estimation methods for both drugs by chromatographic means were lacking until recently. The challenge in developing a simultaneous estimation method lies in the similar physicochemical properties of LID and DIL, particularly their comparable log P (partition coefficient) and pKa values. These similarities make it difficult to achieve adequate separation using chromatographic techniques. Despite this challenge, their structural differences have proven beneficial in achieving resolution between their peaks. A recent study addressed this gap by developing and validating a high performance thin layer chromatographic (HPTLC) method for the simultaneous estimation of LID and DIL, adhering to guidelines outlined in ICH Q2 (R1) [23]. This method not only provides a means to quantify both drugs in a single analysis but also ensures accuracy and reliability in their measurement. The availability of such a method is crucial for pharmaceutical research and clinical practice, enabling efficient formulation development, quality control, and pharmacokinetic studies involving LID and DIL in combination therapies. It represents a significant advancement in analytical chemistry applied to pharmaceuticals, offering a practical solution to the challenges posed by the simultaneous estimation of structurally similar yet functionally distinct compounds like LID and DIL.

2 Experimental

2.1 Standard API, chemicals and materials

The active pharmaceutical ingredients (APIs) of LID and DIL were obtained as complimentary samples from reputable pharmaceutical companies based in Gujarat, India. The marketed gel formulation, known as Crema-L gel, containing 2% w/w of Lidocaine HCl and 2% w/w of Diltiazem HCl, was purchased from a local pharmacy. Chemicals including acetonitrile, toluene, methanol, ethyl acetate, and ammonia were procured from SRL Chemicals Ltd., located in Mumbai, Gujarat.

2.2 Analytical wavelength selection

LID and DIL dissolved readily in acetonitrile solvent. Individual solutions of LID and DIL were prepared at concentrations of 10 µg/ml for analytical purposes. Using a UV–Visible double beam spectrometer (UV-1900i), both solutions were scanned across a wavelength range from 400 to 200 nm. An iso-absorptive point was identified at 220 nm, leading to its selection as the wavelength for quantifying both LID and DIL.

2.3 HPTLC system

The HPTLC setup included a semi-automatic instrument featuring a Linomat 5 sample applicator (CAMAG, Switzerland), a CAMAG TLC scanner IV, and a 100 µl applicator syringe from Hamilton (Switzerland). Chromatographic development was performed using HPTLC aluminium plates sized at 10 cm × 10 cm, pre-coated with silica gel G60-F254 (E. Merck, Darmstadt, Germany; supplied by Anchrom Technologists, Mumbai, India), and twin-through chambers.

2.4 Standard solution preparation

Both LID and DIL were precisely weighed in quantities of 10 mg each and transferred into separate 10 ml volumetric flasks. They were dissolved with a small amount of acetonitrile, and additional acetonitrile was added to achieve a concentration of 1000 µg/ml for each drug individually. Further dilutions were carried out to prepare a mixture containing a concentration of 100 µg/ml for both LID and DIL, which served as the standard working solution.

2.5 Calibration curve determination

Using a Hamilton syringe through the CAMAG Linomat 5 sample applicator, 4 µl, 6 µl, 8 µl, 10 µl, and 12 µl volumes were applied from the standard working solution onto HPTLC silica gel aluminium plates (G60-F254) to construct the calibration curve.

2.6 Assay of marketed formulation

An amount equivalent to 10 mg of LID and DIL from the gel formulation was accurately weighed and transferred into a 10 ml volumetric flask, followed by the addition of a few millilitres of acetonitrile. The mixture was vortexed for ten minutes and then sonicated for another ten minutes. Subsequently, the mixture was filtered through Whatman filter paper No. 45. Acetonitrile was added to achieve a concentration of 1000 µg/ml for both LID and DIL. Further dilution was performed to obtain a concentration of 100 µg/ml of LID and DIL. From the 100 µg/ml solution, 8 µl was applied onto an HPTLC plate using a Hamilton syringe to achieve a concentration of 800 ng/band for LID and DIL. The chromatographic development was carried out up to 85 mm. After development, the plate was removed from the chamber and dried to remove the solvent. Densitometric scanning of the plate was performed at a speed of 20 mm/s using a TLC scanner. The quantities of LID and DIL in the formulation were calculated using regression equations.

2.7 Validation

The developed HPTLC method was validated according to International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH) Q2 (R1) guideline.

2.7.1 Linearity

For calibration curve, the concentrations of 400 ng/band, 600 ng/band, 800 ng/band, 1000 ng/band, and 1200 ng/band were used to ascertain the linear relationship. Plotting peak area vs. concentration revealed a straight-line equation.

2.7.2 Precision

Precision is the degree of accuracy in measurements made from a homogeneous sample. Whereas interday denotes three determinations on separate days, intraday denotes three determinations on the same day. Three concentrations from the linearity range—the lowest, medium, and highest—400 ng/band, 800 ng/band, and 1200 ng/band—were chosen, and calculations were made for intermediate precision. To determine whether or not the procedure is repeatable, 800 ng/band of LID and DIL were used for injection and scanning repeatability. Using the obtained peak areas, the percentage RSD values were computed.

2.7.3 Accuracy

Accuracy is indicated by how closely measured values match their true values. The standard spiking method, in which the standard solutions of LID and DIL were spiked at three levels—80%, 100%, and 120%—is widely used to verify the correctness of the approach. The accuracy was conducted in triplicate, and the percentage of recovery was calculated using the mean of the peak areas.

2.7.4 Limit of detection and quantification

The term “limit of detection” refers to the lowest concentration at which an analyte within a matrix may be detected without quantification. The term "limit of quantification" refers to the lowest concentration at which the analyte may be detected inside the matrix while still being precisely and accurately quantified. The equation used to calculate LOD & LOQ are as shown below.

$${\text{LOD}} = { 3}.{3 } \times \, \sigma /{\text{S }}\& {\text{ LOD}} = { 1}0 \, \times \, \sigma /{\text{S}}$$

where σ is the standard deviation of y- intercepts of regression lines and S is the average slope of calibration curves.

2.7.5 Robustness

Intentionally made minor adjustments to optimal parameters, including run distance, wavelength, chamber saturation time, and mobile phase composition, were made, and results were noted. The method's robustness was evaluated in triplicate at 800 ng/band concentration for both LID and DIL. %RSD values were calculated from mean peak areas.

2.7.6 System suitability

An important part of the method development is the suitability testing of the system to ensure that it is suitable for performing LID and DIL analyses. Before each validation cycle, a system suitability test was performed on the chromatography system. Six identical standard solutions were injected to ensure system suitability. Retention factor (Rf), peak purity and resolution were obtained for each of the six appropriate injections.

2.7.7 Solution stability

Standard solutions were store in refrigerator and estimated to determine stability of solutions of LID and DIL.

3 Results and discussion

3.1 Wavelength selection

The sensitivity of the approach depends on the choice of an appropriate detection wavelength. Standard solutions of LID and DIL (10 µg/ml) were scanned in the UV range of 400–200 nm. UV spectra revealed that both medications absorb noticeably at 220 nm and therefore chosen as the detection wavelength as shown in Fig. 2.

Fig. 2
figure 2

Overlay UV–Visible spectra of Lidocaine HCl and Diltiazem HCl (10 µg/ml)

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3.2 Chromatographic optimization

Pre-coated silica gel G60-F254 aluminium sheet as stationary phase was selected on which 8 mm band length of LID and DIL samples were applied. Various trials were performed to optimize mobile phase which provide good separation between LID and DIL. The final optimized mobile phase obtained was toluene: methanol: acetonitrile: two drops of ammonia (7:2:1 v/v/v). The mobile phase was prepared in volumetric flask by taking appropriate quantities, transferred into 10 × 10 cm camag twin through chamber and kept undisturbed for 30 min for chamber saturation before each chromatographic development. The mobile phase was allowed to migrate about 85 mm distance on HPTLC plate. Then plates are removed and dried to evaporate solvents. Plate was scanned applying 6 × 0.45 mm slit dimension and at speed of 20 mm/s. These optimized conditions gave symmetrical and selective peak for LID at Rf 0.59 and DIL at Rf 0.48 as shown in Fig. 3.

Fig. 3
figure 3

Densitogram of Lidocaine HCl and Diltiazem HCl (800 ng/band)

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3.3 Validation

ICH Q2 (R1) guideline was followed to validate the developed HPTLC method for quantification of LID and DIL simultaneously in bulk and gel dosage form. The results are mentioned and discussed below;

3.3.1 Linearity

LID and DIL were found to be linear within the specified concentration range of 400 ng/band to 1200 ng/band by developed HPTLC method. The overlay of the densitogram shown in Fig. 4. The Table 1 represents the regression analysis result.

Fig. 4:
figure 4

3D Overlay densitogram of Lidocaine HCl and Diltiazem HCl (400–1200 ng/band)

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Table 1 Regression analysis of calibration curve
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3.3.2 Precision

% RSD values for LID and DIL were found to be 0.12–0.47% and 0.36–0.58%, respectively for intraday precision and % RSD values for LID and DIL were found to be 0.16–0.70% and 0.47–0.80%, respectively for interday precision. Lower %RSD values indicate that method is precise for quantifying LID and DIL simultaneously. %RSD for scanning and injection repeatability study was found to be less than 2%. Table 2 displays the result of precision study.

Table 2 Precision data for LID and DIL
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3.3.3 Accuracy

Accuracy study was performed using standard spiking method and % recoveries for LID and DIL were found to be 99.15–101.27% and 99.02–100.59%, respectively. The % recoveries for both the drugs were between the range of 98–102% indicate that the method is accurate. Result of accuracy data has been shown in Table 3.

Table 3 Accuracy study for LID and DIL by HPTLC method
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3.3.4 Limit of detection and limit of quantification

LOD values for LID and DIL were found to be 28.93 ng/band and 18.72 ng/band, respectively. LOQ values for LID and DIL were found to be 87.67 ng/band and 56.74 ng/band, respectively. Lower values indicate that the method is highly sensitive for both detection and quantification of LID and DIL in small quantities.

3.3.5 Robustness

Minor changes in the chamber saturation time, run distance, wavelength, and mobile phase composition were employed for robustness study. For both LID and DIL, no significant change in the Rf values or the bands' compactness were observed. The robustness of method was assessed in triplicate manner at concentration of 800 ng/band for both LID and DIL. % RSD values were found to be less than 2 and data has been shown in Table 4. Thes summary of validation parameters has been shown in Table 5 for the developed HPTLC method.

Table 4 Robustness study for LID and DIL
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Table 5 Result of validation parameter of HPTLC method for Lidocaine HCl and Diltiazem HCl
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3.3.6 System suitability parameters

Resolution between LID and DIL was found to be 2.5. Table 6 represents data obtained from system suitability test.

Table 6 System suitability parameters
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3.3.7 Solution stability

The solution stability data has been shown in Table 7 and it indicates that solutions of LID and DIL were found to be stable up to 24 h.

Table 7 Solution stability
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3.4 Assay of marketed formulation

The presented method was applied for marketed gel formulation to determine LID and DIL, qualitatively as well as quantitatively. The percentage purity for LID was found to be 99.98% ± 0.28 and the percentage purity for DIL was found to be 99.97% ± 1.40. Figure 5 shown the specificity of LID and DIL. Peak purity spectra of LID and DIL has been shown in Fig. 6. The assay result has been shown in Table 8.

Fig. 5
figure 5

Specificity spectra of Lidocaine HCl and Diltiazem HCl

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Fig. 6
figure 6

Peak purity spectra of Lidocaine HCl and Diltiazem HCl

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Table 8 Marketed formulation analysis
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4 Conclusion

The HPTLC method presented in this study offers an advanced yet cost-effective chromatographic approach for the simultaneous determination of LID and DIL. It utilized pre-coated silica gel G60-F254 aluminium sheets as the stationary phase and a mobile phase consisting of toluene: methanol: acetate: 2 drops of ammonia (7:2:1%v/v/v). Detection for both drugs was carried out at 220 nm. The method demonstrated linearity over a concentration range of 400–1200 ng/band for both LID and DIL. The limit of quantitation (LOQ) was determined to be 87.67 ng/band for LID and 56.74 ng/band for DIL. % Recovery studies showed excellent results with LID recovering between 99.15% and 101.27% and DIL recovering between 99.02% and 100.59%. Key advantages of this HPTLC method include its efficiency in solvent usage, allowing multiple analyses in a single run, and its cost-effectiveness compared to traditional liquid chromatographic methods. Furthermore, the method was validated to be precise, accurate, and robust, demonstrating its suitability for the quantitative analysis of LID and DIL in both bulk form and pharmaceutical gel dosage forms. In conclusion, the presented HPTLC method offers a reliable analytical tool for the quantification of LID and DIL, catering to both research and quality control applications in pharmaceutical analysis. Its robust performance and economical advantages make it a practical choice in analytical laboratories.