It is not trivial to generate a chromatographic method with such low uncertainty, but some publication may help [21,22,23,24]. In particular, the ICH guidelines issued by the International Council for Harmonisation of Technical Requirements for Pharmaceuticals [21] are often cited as a quality standard, so that the term “according to ICH guidelines” seems to guarantee the required quality. In this way, ICH guidelines are completely misunderstood, because “the ICH guideline does not provide specific instructions how to validate different analytical methods” [24]. The introduction to guideline Q2(R1) clearly describes its aim: “The objective of validation of an analytical procedure is to demonstrate that the analytical procedure is suitable for the intended purpose.” The purpose of the present work is to demonstrate that the analytical method is suitable for the intended purpose of quantifying trimethoprim and sulfamethoxazole in a pharmaceutical preparation with an acceptable deviation of less than 2.5% and how this can be accomplished.

In chromatography, the analyte is distributed between the mobile and the stationary phase, resulting in an analyte distribution of unknown shape. For quantitative HPTLC, it is not necessary to know the distribution of the analyte in the layer. A constant analyte distribution for all sample and standard zones is essential for quantification. To achieve this, the stationary and mobile phase conditions must be constant for all tracks. As the vapor phase also influences the composition of the mobile phase, which is a result of the solvent distribution in the stationary and vapor phase, the conditions in all three phases must be constant for all analytes with the same RF values during the entire development process [25].

Modern HPTLC should avoid the use of hazardous solvents. However, this is not the case, as shown by the European Pharmacopoeia 10th Edition, where sulfamethoxazole is separated with the mobile phase: dilute ammonia–water–nitromethane–dioxane (3:5:41:51, V/V) [26]. A literature survey reveals that from 13 papers on the analysis of trimethoprim and sulfamethoxazole, 11 papers use CHCl3 as part of the mobile phase [5,6,7,8,9,10,11,12,13,14,15]. Only two publications avoid CHCl3 and instead use a mixture of toluene–ethyl acetate–methanol [4] or a mixture of ethyl acetate–methanol (3:1, V/V), in which trimethoprim unfortunately tends to tail strongly [3].

In the past, we have been successful in replacing CHCl3 by CPME [27], and indeed, a solvent mixture of CPME–methanol (9:2.9, V/V) can separate trimethoprim from sulfamethoxazole (RF = 0.76), with an RF value of 0.16. The disadvantage of this solvent is that trimethoprim shows a large tailing, which makes integration problematic. Sulfamethoxazole moves to a position near the front and peak integration is difficult if the plate is not clean, so a prewash step is mandatory.

The solvent CPME–methanol (9:2.9, V/V) shows that constant phase conditions are present for the trimethoprim peak, which stays close to the application zone. It can be quantified with low uncertainty without chamber saturation being accounted. The sulfamethoxazole zone moves near the front and cannot be quantified with a low uncertainty without chamber saturation. Obviously, the conditions near the solvent surface are constant even in a chamber without a presaturated vapor phase. For the sulfamethoxazole zone at a high RF value, this is not the case, so presaturation of the vapor phase by the solvent is required, using a chamber whose sides are covered with filter paper, which soaks up the solvent and provides a larger surface area for evaporation. It is also helpful to reduce the overall separation distance, and the RF value of the sulfamethoxazole zone can be reduced by preconditioning the plate in the chamber vapor for 10 min.

The solvent mixture CPME–methanol–formamide (8:1.5:0.5, V/V), with the RF values 0.2 and 0.77, avoids tailing of trimethoprim and keeps the trimethoprim band at a larger distance from the application point. Unfortunately, formamide remains entirely in the stationary phase, causing an uneven baseline and making a proper peak integration difficult. The best result was obtained by replacing formamide with water.

In a chamber with a presaturated vapor phase, the solvent mix CPME–methanol–water (7.6:2:0.4, V/V) shows a good separation, no tailing of the peaks, a low RF value of sulfamethoxazole, and a uniform baseline. The densitogram and the structure of both compounds are given in Fig. 2. Both peaks have a sufficiently large distance to the point of application (RF value of trimethoprim is 0.17) and the front signal (RF value of sulfamethoxazole is 0.55). It is important that the plate is prewashed with the solvent methanol–water (8:2, V/V) prior to separation to ensure a uniform baseline.

Densitogram of trimethoprim (left) and sulfamethoxazole (right) on silica gel 60 aluminum foil, separated with the solvent CPME–methanol–water (7.6:2:0.4, V/V)

When selecting the track width of a separation, it must be taken into account that the light fiber width of the diode-array scanner is 3.5 mm. In other words, the sample application must have a homogenous analyte distribution of at least 3.5 mm in length on all tracks. This requires a band-wise application of at least 7 mm. Scanning in the middle of the band allows a constant measurement with uniform sample distribution, even with slight deviations from a perpendicular development. A spot-wise application with slight deviation from a perpendicular development would result in larger scanning deviations. In addition, a band-wise application in sharp bands allows a large application volume without loss of resolution. Keeping a distance of 3 mm to the next band gives track dimensions of 10 mm width.

A CAMAG ATS 4 device was used for band-wise sample application. CAMAG specifies the syringe resolution with 16 bits, thus the program can apply the entire syringe volume in 216 increments. Accepting an application uncertainty of 0.1% for 1 µL (which means a minimum of 1000 steps volume resolution) and using a 500 µL syringe, a rough estimation according to equation (12) shows that this only works if the application volume is larger than 7.6 µL:

$$ \frac}}} }} \ge 7.6\;\upmu } $$

(12)

Application of at least 1.53 µL would provide the required application uncertainty with a 100 µL syringe, as would an application of more than 0.38 µL with a 25 µL syringe.

The stationary phase must have a constant thickness across all tracks. Glass plates (in the size of 10 cm × 20 cm or 10 cm × 10 cm) have a smaller thickness at the plate sides, which is known as the “side effect”. This results in more band broadening at plate sides and, thus, lower analyte concentration in the middle of the band compared with tracks with constant thickness. Therefore, a 10 cm × 10 cm glass plate allows separation of only eight tracks, while on a 10 cm × 10 cm aluminum foil, separation of up to nine tracks is possible.

After separation, the plate must be dried uniformly. During drying of the plate, the analyte in the stationary phase moves towards the plate surface. This increases the analyte concentration at plate surface while the mobile phase evaporates. This is what makes the drying process so important, because uneven drying of the plate will result in different sample distributions on different tracks. It is best to place the plate in a fume hood for 30 min without moving the air above the plate surface. One should never use a hair dryer to dry the plate!

The ICH guidelines state that “specificity/selectivity can be shown by demonstrating that the identification and/or quantitation of an analyte is not impacted by the presence of other substances (e.g., impurities, degradation products, related substances, matrix, or other components present in the operating environment)” [21]. The method presented is selective for trimethoprim and sulfamethoxazole when both compounds are completely separated, and no interfering matrix is present in the analyte zones. To confirm this, a peak-purity check for both compounds is necessary. This is best done using a diode-array scanner. Figure 3 shows the contour plot of a track on which 3.5 µL of a cotrimoxazole sample is separated. The scan was performed in a wavelength range from 200 to 400 nm and shows two symmetrical peaks without wavelength-dependent deformation. The symmetrical structure of both peaks indicates that the separation of trimethoprim, as well as sulfamethoxazole, is not affected by insufficient separation or by the matrix.

Contour plot of a track, separated with the solvent CPME–methanol–water (7.6:2:0.4, V/V) and measured in absorption according to Eq. (4). Trimethoprim and sulfamethoxazole show symmetric peaks

Oxidation of trimethoprim either by dipping in nitric acid [28] or by contact of the plate with oxygen in combination with 24 h daylight irradiation [29] results in a bright fluorescence, which can be used for quantification. Figure 4A shows the contour plot of a cotrimoxazole sample track measured in fluorescence using am LED for illumination that emits light at 365 nm. The fluorescence spectrum of trimethoprim is plotted at left and was evaluated in the wavelength range from 428 to 452 nm by bundling 34 diodes. The resulting peaks of trimethoprim were evaluated, and they showed a standard deviation of always more than 5%. The trimethoprim fluorescence is not suitable for quantification with the required uncertainty.

Contour plot of a track, separated with the solvent CPME–methanol–water (7.6:2:0.4, V/V) and measured in fluorescence (A), according to Eq. (5), and in remission (B), according to Eq. (1). The fluorescence spectrum of trimethoprim in 4A is shown on the left and the densitogram at 440 nm is shown at the top