2.1 Additives, vehicles and materials

All the drugs used are infusion solution concentrates and were obtained from Accord Healthcare Italia S.r.l.: Topotecan 1 mg/mL, Irinotecan 20 mg/mL, Etoposide 20 mg/mL, Doxorubicin 2 mg/mL, Epirubicin 2 mg/mL. 0.9% Sodium Chloride Injection, USP, 100 mL Viaflo Plastic Container (Baxter S.p.A., Rome, Italy); ChemoClave® Universal Vented Vial Spike (Icu Medical California, USA); Microtip Ultra Hypodermic Needle 18 G (Rays S.p.A, Ancona, Italy); monobasic sodium phosphate (GPR, 500 g, BDH Laboratory Supplies, VWR International PBI S.r.l., Milan, Italy); methanol RS for HPLC PLUS Gradient Grade, water HPLC Plus, hydrochloric acid and sodium hydroxide (Carlo Erba Reagents S.a.s., Milan, Italy); sodium hypochlorite (Lombarda H S.r.l, Milan, Italy); Philips UV-C TUV 36 W/G36 T8 (Lampadine professionali, Turin, Italy).

2.2 Instrument and chromatographic conditions

Chromatographic analyses were performed by using the Agilent 1200 HPLC system (Agilent Technologies) equipped with ChemStation revision B.04.01, Degasser G1379B, Binary Pump G1312A, Autosampler (ALS) G1329A, Thermostatted Column Compartment (TCC) G1316A and Diode-Array Detector (DAD) G1315D. Merck Purospher® STAR RP-18 end-capped, 3 µm Hibar® RT 150–4.6 mm column was used for separation. The column temperature was set at 40 °C. The mobile phase consisted in water (adjusted to pH 1.7 with monobasic sodium phosphate buffer 20 mM) and methanol (45/55, v/v) was used for isocratic elution at a flow rate of 1 mL/min. The injection volume was 3 μl. The eluent was monitored at 233 nm for drug detection over a period of 15 min. The method allows for the elution of all drugs within 10 min. The chromatographic conditions are shown in Table 1

2.3 Preparation of samples for validation process

The pharmaceutical specialties were used as a standard for the preparation of all samples as they do not have excipients that absorb at the same wavelength as the drug or that interfere with its spectrophotometric determination (topotecan excipients: tartaric acid, mannitol; irinotecan excipients: sorbitol, lactic acid, sodium hydroxide, hydrochloric acid, water for injections; etoposide: anhydrous citric acid, benzyl alcohol, polysorbate 80, macrogol 300, anhydrous ethanol; doxorubicin and epirubicin excipients: sodium chloride, hydrochloric acid, water for injections) (Peter Wuelfing et al., 2006). All drugs used as raw materials for analysis are in solution in the manufacturer's original vials and each sample was obtained independently by diluting the drug concentrate for solution for infusion with water for HPLC.

All samples were set up using Gilson® pipettes in Agilent glass vials supplied with the HPLC system in use and suitable for the autosampler. After mixing and agitating with the vortex mixer, the sample was visually inspected to verify the absence of precipitates, transparency and correct sample setup (volume matching by comparison with the graduated scale on glass vials). For each type of analysis, the samples were prepared in triple and injected into the column three times (n = 9).

According to the ICH, the concentration range to be analysed for the validation of a method must cover at least 80% to 120% of the test concentration. In our Institute the topotecan in the range of 0.01–0.08 mg/mL, irinotecan in the range of 0.11–1.44 and etoposide in the range of 0.16–0.40 mg/mL are administered intravenously after dilution of the concentrated drug with 0.9% sodium chloride for injection. Doxorubicin and epirubicin are not diluted but administered intravenously at a concentration of 2 mg/mL, for these two drugs the method has been validated at a concentration between 0.02 mg/mL and 0.80 mg /mL since it is planned to analyse their chemical stability by diluting the samples to 0.40 mg/mL before their injection in the HPLC to avoid overloading the column.

The concentrations for the validation protocol (shown in Table 2), were chosen based on these considerations and for the tests in which two different concentrations were analysed, a low and a high concentration were chosen arbitrarily.

2.4 Validation of the HPLC method

After optimising the analytical conditions, linearity, precision, accuracy (recovery), selectivity, limit of detection (LOD), limit of quantification (LOQ) and robustness were evaluated according to the ICH guidelines (The International Conference on Harmonization, 2005). The acceptance criteria considered for the various parameters analysed are those indicated by the Food and Drug Administration (FDA) which in its document “Methods, Method Verification and Validation” (Food and Drug Administration Office of Regulatory Affairs, 2020) summarizes the guidelines contained in ICH and USP.

3.1 Column selection

One of the most important aspects of a chromatographic method with HPLC is to obtain well resolved peaks of the target compounds with respect to any interfering peaks with reasonable analysis times. Fast and efficient analysis is essential when many routine quality control tests need to be performed.

To achieve this goal, all the variables that influence a chromatographic method were evaluated, starting with the choice of the stationary phase. Among the various columns tested (Merck Chromolith HighResolution RP-18 endcapped, Merck SeQuant ZIC-HILIC, Phenomenex Kinetex HILIC, Phenomenex Kinetex C8, Phenomenex Luna Omega PS C18), the one that gave a clear peak resolution between the eluted compounds, sharper symmetric peaks and relatively shorter retention times was the Merck Purospher STAR RP-18.

This column is characterized by high resistance to acids (stability and reproducibility over a wide range of pH 1.5–10.5) and an acid mobile phase was selected to prevent epimerisation of etoposide during the assay (Chow et al., 1987; Lepage et al., 2000) and to have a single ionization state for each drug, thus avoiding doubling, tailing and fronting of chromatographic peaks. To confirm this, looking in the literature it is observed that the other chromatographic methods proposed for these single drugs also involve analysis with acid mobile phases (Bhaskaran et al., 2021; D’Huart et al., 2020; Klasen et al., 2014; Patel et al., 1998; Rodrigues et al., 2009; Sewell and Massimini, 2014; Thiesen and Krämer, 2000).

In addition, the column has excellent stability up to 65 °C and our analysis were made at 40 °C. The high temperature of the column was chosen to shorten the analysis times because at high temperatures the drugs elute earlier, especially doxorubicin and epirubicin, which are those with the longest retention times. Furthermore, at higher temperatures it is possible to work at higher flow rates to accelerate the elution of drugs, without having too high back pressures that could damage the column. Considering that the method involves the use of the water/methanol mixture which has a higher viscosity and therefore gives higher back pressures than the common water/acetonitrile mixture used in chromatography, a high analysis temperature represents an advantage. The use of methanol was preferred over acetonitrile because it is cheaper.

Since the choice of stationary phase depends on the polarity and hydrophilicity/hydrophobicity of the analytes, having 3 of the 5 drugs analysed a negative LogP (topocatecan LogP = -0.535, doxorubicin and epirubicin LogP = -1,899), we tried to use the Hydrophilic Interaction Liquid Chromatography (HILIC) column. This is the most successful approach for the retention and separation of polar compounds. However, even though we had good results in terms of peaks resolution, the analysis had low precision and accuracy, this is a common problem found with the technique other than the reverse phase. Since we needed a reliable method to use in routine quality control analysis, we therefore decided to use the reverse phase which is more unfailing.

3.2 Validation of the HPLC method

The HPLC-UV-DAD method used in our study was fully validated, as shown in the Materials and methods section. The parameters of system suitability are summarised in Table 4. All measured parameters were within the recommended limits according to USP and Ph. Eur.

A typical chromatogram of the mixture of the 5 drugs is shown in Fig. 1. The method effectively separates drugs such as topotecan and irinotecan that belong to the same pharmaceutical class and two epimers such as doxorubicin and epirubicin.

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Fig. 1

Chromatogram and absorbance spectra of topotecan 0.10 mg/mL (tr′ 1.872 min), irinotecan 0.21 mg/mL (tr′ 2.715 min), etoposide 0.21 mg/mL (tr′ 3.663 min), doxorubicin 0.21 mg/mL (tr′ 6.896 min) and epirubicin 0.20 mg/mL (tr′ 8.079 min). tr′ = retention time.

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The specificity of the method was achieved using the UV-DAD detector which clearly showed the absence of interfering peaks in the diluent and of interference between cytotoxic drugs. These results can be seen with chromatograms, the UV profiles and the peak purity of the drugs (Fig. 2).

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Fig. 2

Specificity and Selectivity: chromatogram, absorbance spectra and peak purity of topotecan tr′ 1.956 min (A), irinotecan tr′ 2.675 min (B), etoposide tr′ 3.657 min (C), doxorubicin tr′ 6.554 min (D) and epirubicin tr′ 8.081 min (E). tr′ = retention time.

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The HPLC-DAD method has sufficient sensitivity to determine the drugs under normal conditions of use (diluted or concentrated, stored in vials, bags and syringes) in fact low values ​​of LODs and LOQs were measured, thanks to the proper selection of the detection wavelength, which allowed for low base line noise. Good fitting with the linear model for the response was demonstrated by the analysis of the residuals (data not shown) and by the high value of the determination coefficient (Araujo, 2009), for all the drugs an R² > 0.999 was obtained (Table 5).

The overall bias values ​​obtained were acceptable, in any case the recovery remains in the range of ± 5% (Table 5).

The method was precise, the data of repeatability, intra-day and inter-day variability all had a value of RSDs < 3% (Table 5). Furthermore, the ANOVA test demonstrated the non-significant difference in the area of ​​chromatographic peaks, when the analyses are carried out under the same conditions, both within the whole day and within 8 days (Table 5 and Table 6).

The slight variations in column temperature, mobile phase composition and pH cause changes in the precision and in the bias within the acceptance limit (Table 7). Thus, the developed method can be considered robust.

The degradation analyses have demonstrated the ability of the chromatographic method to discriminate intact drug from degradation products (Fig. 3) and the percentage of drug remaining and retention times after degradation are shown in Table 3. The UV profile of the degradation products observed with the DAD (diode array detector) was different from that of the intact drug. In addition, the peak purity analysis showed that the degradation products did not coelute with drugs (Fig. 2).

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Fig. 3

Forced degradation of topotecan tr′ 1.858 min (A), irinotecan tr′ 2.885 min (B), etoposide tr′ 3.952 min (C) doxorubicin tr′ 6.965 min (D) and epirubicin tr′ 8.191 min (E). UV = ultraviolet light; NaOH = sodium hydroxide; NaClO = sodium hypochlorite; tr′ = retention time.

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Fig. 3

Forced degradation of topotecan tr′ 1.858 min (A), irinotecan tr′ 2.885 min (B), etoposide tr′ 3.952 min (C) doxorubicin tr′ 6.965 min (D) and epirubicin tr′ 8.191 min (E). UV = ultraviolet light; NaOH = sodium hydroxide; NaClO = sodium hypochlorite; tr′ = retention time.

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