Chiral separation of beta-blockers by high-performance liquid chromatography and determination of bisoprolol enantiomers in surface waters

The best results in terms of enantiomer separation were achieved with the Chirobiotic V column and mobile phase consisting of methanol/acetic acid/triethylamine in ratio 100:0.20:0.15 (v/v/v) with flow rate of 0.5 mL/min and column temperature of 45 °C. Table 1 shows the retention factor (k), resolution (R_s), and selectivity factor (α) for each analyte. The retention times of all analytes were within acceptable limits and lasted from 10 to 25 min. Baseline enantiomer separation was achieved with all beta-blockers but labetalol (having two stereogenic centres, i.e. four enantiomers), whose first two enantiomers were only partially separated (Figure 2).

Figure 1

Figure 2

Figure 3

We also tried analysing a mixture of enantiomers of all four beta blockers. The results were good for all analytes but the metoprolol and bisoprolol enantiomers, as one bisoprolol enantiomer co-eluted with one metoprolol enantiomer. We then tried different ratios of acetic acid and triethylamine and different flow rates and column temperatures but none improved the separation in the mixture.

Because of this reason and because bisoprolol is the most common beta-blocker in Croatia and Czech Republic and is the most likely to contaminate surface waters in these countries, we proceeded with method validation for bisoprolol alone.

As the standard solution we used was a racemic mixture, we could not identify the elution order of (R)- and (S)-enantiomers, so we named the first eluted enantiomer BIS1 and the second BIS2. Enantiomeric fractions of BIS1 and BIS2 were 49.7 % and 50.3 %, respectively, confirming the racemic nature of the standard solution.

The IDL of each bisoprolol enantiomer, determined by linear regression, was 0.025 μg/mL for BIS1 and 0.026 μg/ mL for BIS2. These limits were confirmed by injecting standard solution at calculated limit levels (0.025 μg/mL) with S/N ratios higher than 3. The IQL (S/N ratio >10) was 0.075 μg/mL for both enantiomers. These concentrations equal 0.6 μg/L and 1.8 μg/L in surface waters, respectively. Good linearity was achieved for both bisoprolol enantiomers in the concentration range of 0.075–5 μg/mL, with correlation coefficients above 0.998. This range equals 1.8–120 μg/L of each bisoprolol enantiomer in surface waters.

To determine the applicability of this method for bisoprolol enantiomer analysis in real samples, we analysed the solid-phase extracts of surface water samples (taken in Prague and Zagreb) for bisoprolol content. Before solid-phase extraction, however, we added known amounts of standard bisoprolol solution to samples of distilled and surface waters to determine the rate of elution of the analytes from cartridges. These samples were prepared in the same way as regular samples. The recoveries of bisoprolol enantiomers in spiked distilled water at the two concentrations (99–111 %) showed good extraction of bisoprolol.

Samples from the Sava were spiked at three concentrations, 1.80 μg/L (Sava 1), 6.00 μg/L (Sava 2), and 12.00 μg/L (Sava 3). Samples from the Kunraticky stream (Prague 1 and Prague 3) and the nearby pond (Prague 2) were spiked at 12.00 μg/L or lower, according to Table 2. (Figure 3). The recoveries ranged from 100 % to 136 % for enantiomer BIS1 and from 99 % to 138 % for enantiomer BIS2, which is an acceptable range (Table 2). Comparing the recoveries of different samples spiked with 12.00 μg/L of bisoprolol, we can conclude that this method of sample preparation yielded good repeatability, with relative standard deviation of <5 % for both enantiomers.

We also tested the method on surface water samples without spiking them with standard solutions but found no bisoprolol enantiomers. This finding will be addressed further in discussion.

Although the method developed in this study showed better enantiomer separation for each beta-blocker alone than reported previously (1, 9), it failed in separating enantiomers in the mixture of metoprolol and bisoprolol and should perhaps be limited to analysing bisoprolol alone, especially as its consumption and the associated risk of surface water contamination is the highest, at least in Croatia and the Czech Republic. Furthermore, the consumption of metoprolol is not very high in these countries, especially not in Croatia, and its occurrence in surface waters is unlikely, so it is not expected to interfere with bisoprolol enantiomer analysis in surface waters.

Our failure to find bisoprolol enantiomers in unspiked (and therefore real) surface water samples either suggests that bisoprolol is successfully removed in waste water treatment plants or points to another limitation of our method, which is that its detection limits are too high. Further analysis of waste water before and after treatment could give a better insight into the levels of bisoprolol occurring in these countries.

Even with these limitations, however, our method has shown adequate recoveries with spiked samples and could be used to determine bisoprolol enantiomers in surface waters, which, to our knowledge, has not been reported so far. Future research could therefore focus on bisoprolol enantiomer content in waste water collected by treatment plants, where its concentrations may be high. Furthermore, transferring the method to a more sensitive one – such as liquid chromatography coupled with tandem mass spectrometry and using ammonium acetate as the mobile phase additive instead of acetic acid and triethylamine – would perhaps yield much lower limits of detection and quantification (25).

Kiralna separacija beta-blokatora tekućinskom kromatografijom visoke djelotvornosti i određivanje enantiomera bisoprolola u površinskim vodama