RP-HPLC Separation of Acetic and Trifluoroacetic Acids Using Mobile ...

Chem. Anal. (Warsaw), 50, 387 (2005)

RP-HPLC Separation of Acetic and Trifluoroacetic Acids Using Mobile Phase with Ion Interaction Reagent and Without Buffer by Anna Bielejewska1,2* and Bronis³aw K. G³ód3 Pharmaceutical Research Institute, ul. Rydygiera 8, 02–187 Warsaw, Poland 2 Institute of Physical Chemistry, ul. Kasprzaka 44/52, 01–224 Warsaw, Poland 3 Meat and Fat Research Institute, ul. Jubilerska 4, 04-190 Warsaw, Poland 1

Key words:

RP-HPLC, ion interaction reagent (IIR), acetic acid, trifluoroacetic acid, peptide

Ion interaction reagent, IIR, is traditionally added to the mobile phase to improve separation and to increase retention of the ions. Buffer is also added to the mobile phase to stabilize dissociation coefficient. However, it was shown that it is possible to use the mobile phase containing small concentration of IIR without buffer. In the present paper it has been proved that the use of high concentration of IIR without buffer is possible. It enabled us to elaborate a simple, rapid and sensitive RP-HPLC IIR method for the determination of acetic and trifluoroacetic acids (AA and TFA) in synthetic peptides. Separation was carried out using a C18 column and a mobile phase consisting of 4.5 mmol L–1 tetra-nbutylammonium hydrogen sulfate (TBAHS), as IIR, in a mixture of methanol and water (40:60%, v/v). The analytes were determined spectrophotometrically at wavelength of 200 nm. The calibration curves for AA and TFA showed good linearity (the correlation coefficients were better than 0.99 for both compounds) in the concentration range of 2–200 mmol L–1 and 1–10 mmol L–1 for AA and TFA, respectively. Limits of detection (defined as a signal-to-noise ratio 3), were approximately 40 ng (0.03 mmol L–1) and 74 ng (0.05 mmol L–1) for AA and TFA, respectively.

* Corresponding author. E-mail: [email protected]

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W celu polepszenia rozdzielenia i zwiêkszenia retencji jonów tradycyjnie stosuje siê zwi¹zki jonowo-asocjacyjne. Czynnik oddzia³ywuj¹cy z jonami stosuje siê na ogó³ jednoczeœnie z buforem, który stabilizuje stosunek formy zdysocjowanej do niezdysocjowanej analizowanej substancji. Przy zastosowaniu ma³ych stê¿eñ zwi¹zków jonowo-asocjacyjnych u¿ycie buforu nie zawsze jest konieczne. W prezentowanej pracy przedstawiono mo¿liwoœæ zastosowania du¿ego stê¿enia zwi¹zków jonowo-asocjacyjnych bez buforu do oznaczania kwasu octowego i trifluorooctowego w próbkach peptydów syntetycznych. Pomiary prowadzono na fazie C 18 z zastosowaniem fazy ruchomej o nastêpuj¹cym sk³adzie: 4.5 mmol L–1 wodorosiarczanu tetra-n-butyloamoniowego w mieszaninie metanol–woda, 40:60 (v/v). Otrzymane wyniki wykazuj¹ dobr¹ liniowoœæ dla badanych kwasów w zakresie stê¿eñ 2–200 mmol L–1 dla kwasu octowego i 1–10 mmol L –1 dla kwasu trifluorooctowego. Wykrywalnoœæ (zdefiniowana jako trzykrotna wysokoœæ szumów) wynosi dla kwasu octowego 40 ng (co odpowiada stê¿eniu 0,03 mmol L–1) i 74 ng (0,05 mmol L–1) dla kwasu trifluorooctowego.

There is a great need for analytical method, of simultaneously determination of trifluoroacetic (TFA) and acetic acids (AA) in synthetic peptides. In many cases the last step of peptide synthesis is the removal of TFA and protection of the peptide, which after lyophilization from the aqueous acetic acid solution, contains both TFA and AA. Most of the peptides are finally purified using preparative RP–HPLC and gradient elution with acetonitrile and some amounts of TFA. After lyophilization, the pure peptides are obtained as their TFA salts and also contain AA. If synthetic peptides are to be used as drugs, they must be subject to a very careful trifluoroacetate/acetate anions exchange procedure. The completeness of such exchange, i.e. the amounts of AA and TFA in final material must be accurately determined [1–3]. Moreover, volatile fatty acids (for example acetic one) play an important role in food industry, including wine, beer and cheese manufacturing or fruit processing as flavour ingredients [4]. Therefore, the analytical method of determination of AA and TFA in pharmaceuticals and food products is very required. Althought AA and TFA can be separated using ion chromatography [5] (on ion exchange [6] or ion exclusion [7] columns) however they were not separated on the most popular RP–HPLC technique. Generally ions are not separated in reversed phase mode and are eluted in the dead column volume [8]. In pure water, used as a mobile phase, frontal peaks of moderately strong acids are obtained. Additionally their retention depends on solute concentration, what is inconvenient from the analytical point of view. Buffer added to the mobile phase stabilizes dissociation coefficient and, in consequence improves peak symmetry and increases retention. However, it also increases the mobile phase conductivity and therefore the detection limit in e.g. conductometric, electrokinetic, potentiometric is undesirably increased. Ion interaction reagent (IIR) is traditionally added to the mobile phase to improve separation and increase retention of ions. Both, weak electrolytes, including carboxylic acids, as well as strong ones, like sulfonic acids [9], can be separated. The mecha-

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nism of interaction is still doubtful [10]. IIR may form ion pairs with solutes ions, modify the stationary phase as well as influence activity coefficients and dielectric constants of the stationary phase [11]. However, it has been found [12] that small amounts of tetraalkylammonium ions without buffer can improve separation without worsening of detection. Mechanism of this phenomenon is not clear. It was described elsewhere [10] that the increase of the IIR concentration increases hydrophobic adsorption of solutes on the stationary phase and therefore increases also solute retention. However, small concentration of IIR decreases the retention. Probably, in the presence of very small concentration of IIR firstly coats dynamically RP phase and changes retention mechanism to the anion exchange [12]. In the paper we would like to test if it is possible to use IIR at high concentration without buffer and apply it to the simultaneous separation of strong and weak acids. The proposed method was verified by analysing AA and TFA in the sample of synthetic peptides.

EXPERIMENTAL Apparatus Chromatographic experiments were performed using a Shimadzu (Kyoto, Japan) Model LC–10AD VP pump, Rheodyne type 7125 injector (20 mL) and a Schimadzu (Kyoto, Japan) UV VIS detector Model SPD–10AV VP (detection: 200 nm). The column used was: 250 × 4.6 mm I.D. Phenomenex Luna RP 18 and Waters Symetry C18 150 × 4.6 mm ID. The spectra of AA and TFA have been obtained on line using Diode Array Detector SPD M10A VP Schimadzu (Kyoto, Japan) Reagents All reagents (Merck, Darmstadt, Germany and POCh, Gliwice, Poland) were of analytical-reagent grade and were used without further purification. Water was purified using a Millipore (Bedford, USA) Milli–RO4 and Milli–Q water purification systems. Mobile phases were filtered through a 0.45-µm membrane filter (Millipore, Bedford, USA) and degassed under vacuum pump, before used. Leuprolid acetate and synthetic peptide no 200/036A samples were prepared in Peptide Laboratory of the Pharmaceutical Research Institute (Warsaw, Poland). Procedures Chromatographic experiments were performed at flow rate 1 mL min–1. The column was stabilized by passing through it a mobile phase for 1 h prior to the chromatographic separation. The mobile phase consisted of 4.5 mmol L–1 tetrabutylammonium hydrogen sulfate (TBAHS), as IIR, dissolved in water–methanol (60:40, v/v) mixture. All chromatographic studies were performed at the ambient temperature of 20°C in the air-conditioned room.

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In order to elute peptide from the column, the gradient elution was applied: 0–8 min 100% A + 0% B, 8–10 min from 100% to 10% A and from 0% to 90% B, 10–50 min 10% A + 90% B, 50–51min from 10% to 100% A and from 90 to 0% B, 51–60 min 100% A + 0% B. The composition of solvent A was 4.5 mmol L–1 TBAHS in water–methanol mixture (60:40, v/v) and solvent B was methanol of HPLC grade. Stock solutions were prepared by dissolving 0.25 mL of the analyzed acids in 25 mL of Milli–Q water and diluted to the required concentration just before use. 20 mL-samples were injected into the chromatographic system through the Rheodyne injection port using a 100 mL syringe (Hamilton, Reno, NV, USA). The output signal from the photometric detector working at 200 nm was continuously displayed on the chart recorder. Every sample was injected three times and the average of peak areas was taken for further elaboration.

RESULTS AND DISCUSSION Separation Separation of AA and TFA in the conventional RP–HPLC system is difficult due to the significant difference of their pKa values (equal 4.8 and 0.23, respectively). TFA is relatively strong acid and without IIR or with IIR is eluted in the dead volume. The most successful separation was obtained for 4.5 mmol L–1 TBAHS in H2O–MeOH mixture (40:60 v/v). This is presented on Figure 1. It is interesting to note that increase of IIR concentration reversed elution order of the analysed acids, and TFA was retained stronger then AA. Stronger retention of more dissociated TFA suggests that it participates in the ion pair formation.

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Figure 1. Chromatograms of AA and TFA obtained on the column 250 × 4.6 mm I.D. Phenomenex Luna RP–18 with the mobile phase containing of 4.5 mmol L–1 TBAHS, as IIR, in a mixture of methanol and water (40:60%, v/v); flow rate 1 mL min–1; detection at 200 nm

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Influence of pH Retention of acids strongly depends on their dissociation coefficient which depends on the dissociation constant and solute concentration. Both forms of the acid: dissociated and non-dissociated interact differently with the RP support. In fact, only non-dissociated acid is retained on the column. If the mobile phase is unbuffered then both ends of the peak (containing only dissociated acid) move quicker along the column than the peak maximum containing also non-dissociated form of the acid. In conse-quence in the unbuffered mobile phases frontal tailing of the chromatographic peaks is observed [12]. To stabilize dissociation coefficients buffered mobile phases are used. The same ratio of the dissociated to non-dissociated form of the solute is maintained. This ratio is responsible for the peak symmetry. In the reversed phase chromatography lowering of the mobile phase pH results in a decreased dissociation of the acidic solute and, in consequence, increases retention.

   

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Figure 2. Chromatograms of TFA obtained on the column 250 × 4.6 mm I.D. Phenomenex Luna RP–18 with the mobile phase consisting of: A) 4.5 mmol L–1 TBAHS, as IIR, in a mixture of methanol and water (40:60%, v/v) and B) 4.5 mmol L–1 TBAHS, as IIR, in a mixture of methanol and water (40:60%, v/v) with 0.7 mL L –1 H3PO4 pH 3 (NaOH)

In this study was checked how the addition of phosphorous acid together with ionpairing reagent to the mobile phase affects the retention and the shape of chromatographic peaks. In Figure 2 two chromatograms, obtained in the presence of and without acid are presented. Any significant difference between the two chromatograms was observed. This means that interactions between IIR and deprotonated form of the acid facilitates its dissociation.

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Linearity The linear dynamic range of the peak area of AA was 1–200 mmol L –1 (R = 0.995). For TFA upper limit of the linear dynamic range was lower and equal aprox. to 10 mmol L–1. At lower concentration (< 10 mmol L–1) symmetrical, gaussian peaks of TFA were obtained (Fig. 1). The increased concentration above that of IIR causes that not all TFA molecules participate in the formation of ion pairs. As a result frontally tailing peaks were obtained, similar to those observed for acids separated in pure water (Fig. 3 – 10 mmol L–1). Further increase of TFA concentration resulted in double peaks, which corresponded to two forms of the acids: dissociated and non-dissociated (Fig. 3 – 100 mmol L–1). 2

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Detection and determination levels The analyzed acids were characterized by similar spectra. In the spectrum of TFA a very narrow maximum at 200 nm can be distinguished. Detection level of TFA strictly depends on the detection wavelength. The wavelength of 200 nm was chosen as optimum for both acids, regarding their detection limits which equal to 40 ng (0.03 mmol L–1) and 74 ng (0.05 mmol L–1) for AA and TFA, respectively.

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Comparison with European Pharmacopeia 4 procedure for AA determination The results of AA assay were compared to those obtained using the method in the European Pharmacopoeia 4. In Figure 4 chromatograms of AA obtained for different mobile phases are presented. Compared to other methods, our approach provides better performance and efficiency, as well as lower detection limit.  

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Figure 4. Chromatograms of 2.5 mmol L–1 AA obtained on the column 150 × 4.6 mm I.D. Waters Symetry with the mobile phase containing of A): 4.5 mmol L–1 TBAHS, as IIR, in a mixture of methanol and water (40:60%, v/v), and B): H20 with 0.7 mL L–1 H3PO4 pH 3 (NaOH–MeOH, 95:5% v/v) according to the European Pharmacopoeia 4

Practical application The described method was applied to the determination of AA and TFA in synthetic peptides: leuprolide acetate and peptide No 200/036A. Concentration of AA in both studied samples of peptides was about 6%, the TFA was found only in the sample No 200/036A at the concentration level 0.9%. In Figure 5 chromatograms of leuprolide samples spiked by AA and TFA are presented.

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Figure 5. Chromatogram of leuprolide samples spiked by AA and TFA. Chromatographic conditions: column 250 × 4.6 mm I.D. Phenomenex Luna RP-18, flow 1 mL min–1, the gradient elution was: 0–8 min 100% A, 0% B, 8–10 min from 100% to 10% A and from 0% to 90% B 10–50 min 10% A, 90% B, 50–51 min from 10% to 100% A and from 90 to 0% B, 51–60 min 100% A, 0%B the composition of solvent A was 4.5 mmol L–1 TBAHS in water–methanol (60:40, v/v) mixture and solvent B was methanol for HPLC; detection at 200 nm

CONCLUSIONS It was shown that AA and TFA can be analysed simultaneously using IIR without buffer. The calibration plots for AA and TFA revealed good linearity (the correlation coefficients were better than 0.99 in both cases) in the concentration ranges 2–200 mmol L–1 and 1–10 mmol L–1 for AA and TFA, respectively. Detection limits, at the wavelength 200 nm (defined as a signal-to-noise ratio equals 3), were approximately 40 ng and 74 ng for AA and TFA, respectively. The new method is characterized by better performance and efficiency, as well as lower detection compared to the method described in Pharmacopoeia for the analysis of AA in peptide samples. Acknowledgements We would like to thank Dr. Krzysztof Bañkowski for providing the samples of peptides and for his valuable input.

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Received July 2004 Accepted August 2004