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August/September 2011
LC-MS/MS instrument. Instead of processing 1 L of water by the manual, time consuming process of SPE described in EPA Method 1694, this alternative approach incorporates online sample preparation in series with LC- MS/MS using smaller volumes of water (0.5 - 20 mL) to achieve ng/L quantitation limits. This article will demonstrate a progressive approach to analysing PPCPs in environmental sources of water at the ng/L level with online sample preparation using small volumes of water, thus saving time and reducing the cost of analysis
Experimental Water samples of 0.5 mL were directly injected onto a pre-concentration trapping
column (2.1 x10 mm, 12 µm) at 1.5 mL/mins with H2O + 0.2% formic acid. After sufficient washing of the pre-concentration column, the target compounds were transferred to
the analytical column (2.1 x 100 mm, 3 µm) for chromatographic separation by gradient elution prior to introduction into the mass spectrometer.
MS analysis was carried out with a Thermo Scientific TSQ Vantage triple stage quadrupole mass spectrometer. Two selected reaction monitoring (SRM) transitions per compound were acquired; one for quantitation with the other for positive confirmation. To maximise the performance of the triple stage quadrupole, time-specific SRM ‘windows’ were employed at the retention times of the target compounds.
Results and Discussion The current EPA method 1694 describes three different LC methods for PPCPs from Groups 1, 2 and 4 which are amenable to positive electrospray ionisation (ESI) MS/MS. In order to simplify the method and reduce the total analysis time, a single 10-minute LC- MS/MS method was developed which includes compounds from additional pharmaceutical classes not included in EPA Method 1694, such as beta-blockers. In total, 67 compounds were analysed by positive ESI-MS/MS (see Table 1). Of these, 54 were from EPA Method 1694 Groups 1, 2 and 4.
Such a diverse range of chemical classes meant the challenge was in developing a single LC-MS/MS method without compromising the target ng/L sensitivity. Both sample pH and the %ACN in the sample influenced the response of PPCPs in water when employing the online sample preparation approach with the EQuan system. To determine the best method for
Figure 2. Area response plots demonstrating the pH effect on the sample solubility.
Figure 1. Chromatograms showing the pH effect on chlorotetracycline (CTC).
achieving ng/L sensitivity on the mass spectrometer, the effects of sample pH and %ACN were investigated.
Effects of Sample pH It was found that sample pH affected the response of some PPCPs in water based on chemical reactivity. During the method development, PPCPs were added to aqueous solutions at three different pHs: 2.9, 6.6 and 11.3. As shown in the chromatograms in Figure 1, chlortetracycline (CTC) was readily observed at pH 2.9 and pH 6.6. However, at pH 11.3, CTC completely disappeared, being converted to 4-epi-CTC. It is important to note that no 4-epi-CTC was added to the water samples prior to LC- MS/MS analysis. All of the 4-epi-CTC detected was a result of the conversion of CTC, which has been shown to have a short half life in solutions at pH 11.2. A similar effect was observed with erythromycin, which reacted quickly in acidic solution and converted to anhydroerythomycin at pH 2.9.
The pH also affected the solubility of some PPCPs, even within the same compound
class. Figure 2 displays the area response for cloxacillin and penicillin. For cloxacillin, the area response at pH 6.6 is evident in the bar chart at the top left; whereas at pH 11.3, cloxacillin was not observed. A similar effect was seen for ampicillin, oxacillin, cefotaxime and diltiazem. However, the opposite effect was observed for penicillin V (and G), as seen in the bar chart. The same trends were also
observed with LC-MS/MS (5.0 µL injection) as with the EQuan method (0.5 mL injection) indicating that this is a sample solubility effect.
The pH effect on the MS response was also observed with several other PPCPs when using the EQuan system. Using ranitidine as an example, the MS response was observed with several other PPCPs when using the system. The MS response was much greater at pH 11.3 than at pH 2.9 or 6.6, as shown in the chart at the top left of Figure 3. However, this pH effect was not present when using a
5.0 µL injection of the water samples directly onto the analytical column at the same mass loading of ranitidine, as seen in the bar chart in the lower right of Figure 3. This difference
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