Application of isotachophoretic and conductometric methods for

Chemical Papers 63 (3) 255–260 (2009) DOI: 10.2478/s11696-008-0103-2

ORIGINAL PAPER

Application of isotachophoretic and conductometric methods for neomycin trisulphate determination Marzanna Kurzawa*, Aneta Jastrz˛ ebska, Edward Szlyk Faculty of Chemistry, Nicolaus Copernicus University, 87-100 Toru´ n, 7 Gagarin str., Poland Received 18 January 2008; Revised 3 September 2008; Accepted 4 September 2008

Determination of neomycin trisulphate (NMS) in a dosage form (Neox and Neosol) was carried out by capillary isotachophoresis (cITP) with conductometric detection. The following electrolytes: leading: 10 mmol dm−3 sodium acetate + 0.08 % hydroxyethylcelulose (HEC) and acetic acid to pH = 5.5, and terminating: 10 mmol dm−3 β-alanine were tested for isotachophoretic separation of NMS. The calibration curve was linear over the range of 10.00 mg dm−3 to 100.00 mg dm−3 with LOD = 5.69 mg dm−3 and LOQ = 18.96 mg dm−3 . The results were compared to the conductometric determination of NMS with: ammonium molybdate (VI), silver nitrate (V) and Reinecke salt. Good accuracy was obtained from conductometric titration of NMS with Reinecke salt, the recoveries being as follows: 100 % (RSD = 1.99 %); 96.17 % (RSD = 2.10 %) and 95.22 % (RSD = 1.55 %) for NMS in pure form, Neosol and Neox, respectively. c 2008 Institute of Chemistry, Slovak Academy of Sciences Keywords: capillary isotachophoresis, neomycin trisulphate, Neox, Neosol, conductometric titration

Introduction Neomycin, belonging to aminoglycosides, is used as an antibiotic in the treatment of animals because of its wide spectrum of activity against gram-negative bacteria (British Pharmacopoeia, 2001). However, this compound is harmful and it must not enter human food. Therefore, long withdrawal periods are required before dosed animals may be slaughtered. Hence, the analysis of this antibiotic is important for the food industry. Various chromatographic methods are commonly used for neomycin trisulphate (NMS) determination. Posyniak et al. (2001) applied liquid chromatography (LC) using a reversed-phase column with fluorimetric detection for the quantification of NMS and gentamycin in animal tissues. The calibration curves were linear from 0.2 mg kg−1 to 1.0 mg kg−1 with the limits of detection (LOD) at 0.10 mg kg−1 . Recoveries of neomycin spiked at levels of 0.2 mg kg−1 porcine tissues ranged from 77 % to 83 %. A specific and automated chromatographic method was developed for *Corresponding author, e-mail: [email protected]

the assay of neomycin in human serum by Oertel et al. (2004). Neomycin was also detected with electrospray ionisation tandem mass spectrometry (ESI–MS– MS). The correlation coefficient of regression lines was 0.9985 or higher, the accuracy ranging from 95.7 % to 104.2 %. The novel HPLC–ELSD (evaporative light scattering detection) method for the determination of neomycin sulphate was applied by Megoulas and Koupparis (2004) (LOD 0.6 mg dm−3 and RSD = 1.7 %). The developed method was also applied for the determination of neomycin in pharmaceutical formulations (powder, aerosol and cream) with recovery ranging from 99 % to 102 % and RSD values being less than 2.1 %. On the other hand, this technique requires modern and expensive instrumentation with high purity reagents; this is why a much simpler technique is still desired. Capillary isotachophoresis (cITP) was tested as a complementary method to HPLC in the determination of pharmaceutical preparations (Polášek et al., 2000; Pospíšilová et al., 1997, 1998; Valášková et al., 1995; Hernandez et al., 2002). Furthermore, Klein and Teichmann (1982) described isotachophoretic assay of

256

M. Kurzawa et al./Chemical Papers 63 (3) 255–260 (2009)

aminoglycosides and lincomycines using UV and conductometric detectors. The authors applied the following electrolytes system: leading (LE) – 20 mmol dm−3 potassium acetate and 0.3 % hydroxymethylpropylcellulose (pH = 4.95), and terminating (TE) – 20 mmol dm−3 1,4-aminobutyric acid and acetic acid (pH = 4.72), or 20 mmol dm−3 glycylglycine, or 20 mmol dm−3 β-alanine. The obtained results confirmed the possibility of using capillary isotachophoresis (cITP) for the determination of these drug classes at the 1.6 nmol level with recoveries ranging from 91.6 % to 109.2 %. An isotachophoretic method coupled with conductivity detection was developed to determine naproxen in human serum (Čakrt et al., 2001), as well as ibuprofen and naproxen in tablets (Sádecká et al., 2001). The calibration curves revealed linearity from 0.4 mg dm−3 to 2.4 mg dm−3 for ibuprofen and from 5.0 µg dm−3 to 20.0 µg dm−3 for naproxen. The application of cITP for the determination of fenoprofen in serum at the concentration levels from 0.02 mmol dm−3 to 0.4 mmol dm−3 with RSD in the range of 0.001–0.004 % was described by Sádecká et al. (1999). Sádecká and Polonský (1996) applied capillary isotachophoresis for the determination of some cardiovascular drugs in serum and urine reporting an LOD range from 32 ng cm−3 to 46 ng cm−3 of the drug in urine and from 39 ng cm−3 to 46 ng cm−3 in serum with the recoveries from 98.2 % to 103.2 %. In our previous paper (Kurzawa et al., 2005), an indirect method for NMS determination in form of sulphates resulting in a good separation of sulphates in the composed matrix of the drug Enterogast was proposed. On the other hand, no literature reports concerning the determination of neomycin sulphate as the neomycin cation by cITP were found. For this reason, the main goal of the presented work is an application of cITP for direct determination of NMS in pure form and in two veterinary preparations (Neox and Neosol). A simple electrolytes system was applied: leading electrolyte (LE) – 10 mmol dm−3 sodium acetate, 0.08 % HEC and acetic acid to reach pH = 5.5, and terminating electrolyte (TE): 10 mmol dm−3 β-alanine (BALA). The cITP results were compared with those obtained from conductometric titration using three different titrants (ammonium molybdate (VI), silver nitrate (V), Reinecke salt). The results obtained were discussed in respect to their accuracy and precision.

crobiological method), Neosol (Neomycin trisulphate – 145 g and vehiculum to 1000 cm3 ) and Neox (Neomycin trisulphate – 1.5 g, oxytetracycline hydrochloride – 1.5 g and vehiculum to 100 cm3 ) were obtained from Biowet (Gorzów Wlkp., Poland). Redistilled water (specific conductivity below 2 µS cm−1 ) was used for sample solution preparation. The conductivity measurements were carried out with a conductivity meter CX-742 (Elmetron, Poland) using the bell-shaped electrode EPS-2ZM type (Radelkis). Isotachophoretic separations were performed using a Villa Labeco EA 100/101 isotachophoretic analyser equipped with a conductivity detector. A PTFE pre-separation capillary (90 mm × 0.8 mm I.D.) was connected with a PTFE analytical capillary (160 mm × 0.3 mm I.D.). Samples were injected via a sample valve of 30 µL fixed volume by an internal sample loop. Isotachopherograms were evaluated using the PC software package supplied with the analyser (KasComp, Slovakia). Isotachophoretic conditions: Neomycin trisulphate was analysed by isotachophoresis using the experimental conditions as follows: leading electrolyte (LE) – 10 mmol dm−3 sodium acetate + 0.08 % HEC and acetic acid to reach pH = 5.5, and terminating electrolyte (TE) – 10 mmol dm−3 β-alanine (BALA). The driving current of the pre-separation column was 250 µA. Conductometric titration: Concentration of the antibiotic solution used in conductometric titrations was 4.993 × 10−4 mol dm−3 . As titrants 5.001 × 10−3 mol dm−3 (NH4 )6 Mo7 O24 , 5.214 × 10−3 mol dm−3 AgNO3 , and 6.561 × 10−2 mol dm−3 NH4 [Cr(NCS)4 (NH3 )2 ] were used. All experiments were carried out in aqueous solutions at room temperature (298 ± 2) K. An aliquot of 15 cm3 or 20 cm3 (depending on the titrant) of the drug was placed in the conductometric vessel and titrated using the above solutions. For equilibration of the reaction, 0.1 cm3 (or 0.2 cm3 , depending on the sample concentration) of a titrant was added, stirred for 2 min and left for another 2 min. Titrations were repeated three times and the average values were calculated. After the addition of the titrants, precipitates were formed.

Experimental

Neomycin was identified using the relative step height (RSH) parameter calculated from the relation: RSHX = (HX – HL)/(HT – HL), where HX is the zone height of the NMS cation, HL and HT are step heights of the leading and terminating ion, respectively (Everaerts et al., 1976). The first derivative method was used for better qualitative and quantitative analyses of NMS in pharmaceuticals. A standard stock solution of neomycin trisulphate (100 mg dm−3 ) in redistilled water was used to pre-

Materials and methods Ammonium molybdate(VI), silver nitrate(V), acetic acid and sodium acetate (all of analytical grade) were purchased from POCH Gliwice (Poland), while Reinecke salt, β-alanine (BALA) and hydroxyethylcellulose (HEC) were purchased from Sigma–Aldrich. Neomycin trisulphate (95.20 %, determined by a mi-

Results and discussion Isotachophoretic determination

257


Table 1. Zone length (L) and relative step height (RSH) values for neomycin trisulphate at different concentrations (n = 5) NMS concentration

Zone length

RSDRSH within-day

RSDRSH between-days

%

%

4.67 3.25 2.85 2.47 2.38 2.53

5.01 3.27 2.75 2.54 2.41 2.53

RSH

mg dm−3

s

10 30 50 70 90 100

52.00 62.82 73.90 88.97 100.75 106.90

0.0150 0.0154 0.0158 0.0162 0.0168 0.0158

2.2

0.4

a

b

0.3 1.8

U/V

U/V

0.2

1.4

NMS

1.0 415 435 455 475 495 515 535 555 Time/s

0.1 0 -0.1 415 435 455 475 495 515 535 Time/s

Fig. 1. Isotachopherogram for neomycin trisulphate (50 mg dm−3 ) (a) with the first derivative (b). Isotachophoretic conditions: LE – 10 mmol dm−3 sodium acetate + 0.08 % HEC and acetic acid to pH = 5.5 and TE – 10 mmol dm−3 BALA; I = 250 µA.

pare working solutions. Six working solutions of NMS with the concentration ranging from 10 mg dm−3 to 100 mg dm−3 were used for calibration. Withinday analyses were carried out by injection of the NMS standard solutions (five times per day). Intralaboratory reproducibility (between-days) was determined by analysing the standard solutions (on five different days). Results obtained are listed in Table 1. RSH values ranging from 0.0150 to 0.0168 in the studied concentrations of NMS were quite stable. Effective separation was achieved with the proposed buffer system at pH 5.5. A typical isotachopherogram of NMS at the concentration of 50 mg dm−3 is presented in Fig. 1. The RSD values of the zone length were below 4.67 % (n = 5), which reveals a high reliability and within-day precision. Furthermore, RSD for betweendays precision indicated satisfactory intra-laboratory reproducibility and stability of the proposed electrolyte system. The calibration curves for the concentration range from 10 mg dm−3 to 100 mg dm−3 were established for the quantitation of NMS. The calibration curve expressed as y = (b ± Sb )x + (a ± Sa ), where Sb , Sa are standard deviations of slope and intercept, respectively, resulted in the regression equation: y = (0.6203 ± 0.0150)x + (44.706 ± 1.002) with linear regression values r2 = 0.9976. The limit of detection (LOD) of NMS was calculated from (y +3sy/x)/b, where the cal-

culated intercept of the calibration curve can be used as an estimate of y, sy/x being the standard deviation in the y-direction of the calibration curve and b being the slope of the calibration curve. The 10 sy/x /b expression was used to estimate the quantification limit, LOQ (Miller & Miller, 2000) resulting in LOD and LOQ 5.69 mg dm−3 and 18.96 mg dm−3 , respectively. The elaborated method of neomycin trisulphate determination was tested on the pharmaceutical preparations Neox and Neosol at the following concentrations: 50 mg dm−3 , 70 mg dm−3 , 90 mg dm−3 , and 100 mg dm−3 . The typical isotachopherograms are presented in Figs. 2 and 3. The analyses were performed under the same conditions as the calibration curve, the results being listed in Table 2. The accuracy, expressed as recovery, for the studied preparations depends on the type of sample and NMS concentration in the studied pharmaceutical. Higher recoveries were obtained for Neosol, probably due to the lack of interfering substances in the vehiculum. The recoveries of NMS concentration at the levels: 70 mg dm−3 , 90 mg dm−3 , and 100 mg dm−3 were ≥ 95.47 %; this indicated satisfactory accuracy for routine analysis of the active substance in pharmaceutical preparation (Green, 1996). Furthermore, the precision calculated as RSD values can be acceptable in pharmaceutical analysis. Unfortunately, in the case of Neox, the obtained recoveries were lower (≤ 94.90 %), which was probably caused by complex

258


0.4

3.0

b

a

0.3

U/V

U/V

2.6 2.2 NMS

0.2

1.8

0.1

1.4

0 -0.1

1. 0 125

175

225 275 Time/s

325

125

375

175

225 275 Time/s

325

375

Fig. 2. Isotachopherogram of Neosol (concentration of NMS – 50 mg dm−3 ) (a) with the first derivative (b). Isotachophoretic condition: LE – 10 mmol dm−3 sodium acetate + 0.08 % HEC and acetic acid to pH = 5.5 and TE – 10 mmol dm−3 BALA; I = 250 µA.

0.4

3.3 a

0.3

b

U/V

U/V

2.8 NMS

2.3

0.1 0

1.8 1.3 210

0.2

-0.1

260

310

360

410

460

210

260

310

360

410

460

Time/s

Time/s

Fig. 3. Isotachopherogram for Neox (concentration of NMS – 50 mg dm−3 ) (a) with the first derivative (b). Isotachophoretic condition: LE – 10 mmol dm−3 sodium acetate + 0.08 % HEC and acetic acid to pH = 5.5 and TE – 10 mmol dm−3 BALA; I = 250 µA. Table 2. Determinations of neomycin trisulphate in pharmaceuticals (Neox and Neosol) by isotachophoresis (n = 5) Calculated/(mg dm−3 )

Found (x ± µ)/(mg dm−3 )

RSD/%

Recovery/%

6.11 2.43 1.84 1.88

83.84 94.03 94.90 93.58

4.71 3.01 1.19 1.54

91.70 95.47 97.77 97.07

Neox 50 70 90 100

41.92 65.82 85.43 93.58

± ± ± ±

3.18 1.99 1.95 2.19

Neosol 50 70 90 100

45.85 66.83 87.66 97.07

± ± ± ±

2.69 2.50 1.29 1.86

(x ± µ) is the average value with confidence limit (p = 95 %); n the number of samples; RSD the relative standard deviation.

constitution of Neox which contains, besides neomycin trisulphate, also oxytetracycline hydrochloride and vehiculum. The addition zones were observed on isotachopherograms (Fig. 3). These observations suggest that the proposed electrolyte system is adequate for the determination of NMS in pure form but not for complex pharmaceuticals. It can be noted that the proposed electrolyte

system allows for the use of a pre-separation column for the separation and determination of NMS, while the time of analysis is short (< 9 min). It can be concluded that this electrolyte system can be successfully applied to routine analysis of a single drug (neomycin). Unfortunately, for combined drugs, cITP did not lead to satisfactory results unless a modifier or a complexing agent was introduced into the leading electrolyte

259


Table 3. Determination of neomycin trisulphate in pure form and in pharmaceuticals (Neosol, Neox) by conductometric titration (n = 5) Neomycin trisulphate

Neosol

Neox

14.50 13.62 ± 0.63 1.87 93.93

4.50 4.36 ± 0.28 2.61 96.88

10.87 10.05 ± 0.43 1.71 92.46

4.50 4.40 ± 0.10 0.93 97.78

13.05 12.55 ± 0.66 2.10 96.17

9.00 8.57 ± 0.33 1.55 95.22

ammonium molybdate (VI) Sample mass/mg Found (x ± µ)/mg RSD/% Recovery/%

6.48 6.29 ± 0.24 1.56 97.07 silver nitrate (V)

Sample mass/mg Found (x ± µ)/mg RSD/% Recovery/%

6.48 6.40 ± 0.20 1.25 98.77 Reinecke salt

Sample mass/mg Found (x ± µ)/mg RSD/% Recovery/%

12.65 12.65 ± 0.62 1.99 100.00

in order to increase the difference of electrophoretic mobilities.

0.8

y = 0.3571x – 0.1394

0.6

Results obtained by the cITP method were compared with conductometric titration. Previously, this technique was successfully applied for the determination of phenothiazines and butyrophenones (Kurzawa et al., 2004; Kowalczyk-Marzec et al., 2002). For NMS determination, three different titrants (ammonium molybdate (VI), silver nitrate (V), Reinecke salt) were applied. The obtained results for neomycin trisulphate in pure form suggest that the reaction proceeded in the mole ratios as follows: 1 : 1 with (NH4 )6 Mo7 O24 ; 1 : 6 with AgNO3 and 1 : 6 with NH4 [Cr(NCS)4 (NH3 )2 ]. This indicates that only H2 SO4 (connected with neomycin) takes part in the reaction with titrants: C23 H46 N6 O13 · 3H2 SO4 + (NH4 )6 Mo7 O24 → → [C23 H46 N6 O13 · 6H+ ] · [Mo7 O6− 24 ]↓ + + 3(NH4 )2 SO4

(1)

C23 H46 N6 O13 · 3H2 SO4 + 6AgNO3 → → C23 H46 N6 O13 · 6HNO3 + 3Ag2 SO4 ↓

(2)

C23 H46 N6 O13 · 3H2 SO4 + 6NH4 [Cr(NCS)4 (NH3 )2 ] → → [C23 H46 N6 O13 · 6H+ ] · [Cr(NCS)4 (NH3 )+ 2 ]6 ↓ + + 3(NH4 )2 SO4 (3) The product of a reaction between NMS, ammonium molybdate (VI) and Reinecke salt is probably in form of an ionic-pair. The end-point in the conductometric titration was detected by extrapolation of two linear branches of the

χ/mS

Conductometric determination of NMS

0.4

0.2

y = 0.1368x + 0.1603 0 0

0.5

1

1.5

2

2.5

V/mL

Fig. 4. Conductometric titration curve of neomycin trisulphate with ammonium molybdate (VI).

plot. The shapes of titration curves were typical for this type of measurements, being similar for all studied titrants. The titration curve of neomycin trisulphate by ammonium molybdate (VI) is presented in Fig. 4. Results of conductometric determination of neomycin trisulphate in pure and dosage form (Neosol, Neox) are listed in Table 3. The recovery values obtained for conductometric determination of neomycin trisulphate in pure form are satisfactory for pharmaceutical analysis (Miller & Miller, 2000; Green, 1996), while the best titrant appears to be Reinecke salt (recovery 100 %). Unfortunately, the precision of the latter titrant, expressed as RSD, was the lowest but still satisfactory. In order to prove the usefulness of the elaborated method, determination of neomycin in pharmaceutical preparations (Neox and Neosol) was carried out in a way similar to

260


the procedure described for the pure form of the antibiotic. Neomycin determination in Neosol resulted in recovery values below 100%, ranging from 92.46 % (RSD = 1.71 %) for titration with silver nitrate (V) to 96.17 % (RSD = 2.10 %) with Reinecke salt (analogically as with the antibiotic in pure form). The recovery values obtained for conductometric titration of Neox ranged from 95.22 % (Reinecke salt) to 97.78 % (AgNO3 ). The lowest RSD value was obtained for the titration with AgNO3 (0.93 %). The lower recovery values obtained for this pharmaceutical preparation may result from the influence of oxytetracycline and vehiculum.

Conclusions The proposed methods (cITP and conductometric titration) allow for the determination of NMS in pure form and in some pharmaceutical formulations. Both methods exhibit a similar value of accuracy, whereas the precision of conductometric titration is better. Moreover, the recovery values obtained by the elaborated methods were compared by statistical tests (tStudent and f-Snedecor). In the case of Neosol, both methods are comparable in respect to their precision and accuracy. For Neox, the null hypothesis was retained only for the cITP results compared to the titration by ammonium molybdate (VI) with no significant differences between both methods. Sufficient sensitivity and accuracy values, low running costs and lower consumption of organic solvents are in favour of the cITP technique as an alternative to the commonly used chromatographic methods. Furthermore, this technique is less time-consuming than conductometric titrations. In conclusion, the isotachophoretic method can be applied for the determination of the studied antibiotic in pure form for routine quality control analysis. References British Pharmacopoeia. (2001). London: The British Pharmacopoeia Commission. Retrieved from http://www. pharmacopoeia.co.uk/. Čakrt, M., Hercegová, A., Leško, J., Polonský, J., Sádecká, J., & Skačáni, I. (2001). Isotachophoretic determination of naproxen in the presence of its metabolite in human serum. Journal of Chromatography A, 916, 207–214. DOI: 10.1016/S0021-9673(00)01071-2. Everaerts, F. M., Beckers, J. L., & Verheggen, T. P. E. M. (1976). Isotachophoresis, theory, instrumentation and applications. Amsterdam: Elsevier. Green, J. M. (1996). A practical guide to analytical method validation. Analytical Chemistry, 68, 305A–309A. Hernandez, M., Aguilar, C., Borrull, F., & Calull, M. (2002). Determination of ciprofloxacin, enrofloxacin and flumequine in pig plasma samples by capillary isotachophoresis–capillary zone electrophoresis. Journal of Chromatography B, 772, 163–172. DOI: 10.1016/S1570-0232(02)00071-5.

Klein, H., & Teichmann, R. (1982). Isotachophoretic assay of aminoglycosides and lincomycins in pharmaceuticals. Journal of Chromatography, 250, 152–156. DOI: 10.1016/S00219673(00)95228-2. Kowalczyk-Marzec, A., Kurzawa, M., Szydlowska-Czerniak, A., & Szlyk, E. (2002). Conductometric determination of phenothiazine derivatives by precipitation titration. Chemical Analysis (Warsaw), 47, 613–618. Kurzawa, M., Kowalczyk-Marzec, A., & Szlyk, E. (2004). Conductometric and spectrophotometric determination of haloperidol, droperidol and pimozide. Chemical Analysis (Warsaw), 49, 91–99. Kurzawa, M., Jastrz˛ebska, A., & Szlyk, E. (2005). Indirect determination of neomycin trisulphate as sulphates by column coupling capillary isotachophoresis. Acta Poloniae Pharmaceutica, 62, 163–169. Megoulas, N. C., & Koupparis, M. A. (2004). Enhancement of evaporative light scattering detection in high-performance liquid chromatographic determination of neomycin based on highly volatile mobile phase, high-molecular-mass ion-pairing reagents and controlled peak shape. Journal of Chromatography A, 1057, 125–131. DOI: 10.1016/j.chroma.2004.09.052. Miller, J. N., & Miller, J. C. (2000). Statistics and chemometrics for analytical chemistry (4th ed.). Harrow, U.K.: Pearson Education/Prentice Hall. Oertel, R., Renner, U., & Kirch, W. (2004). Determination of neomycin by LC–tandem mass spectrometry using hydrophilic interaction chromatography. Journal of Pharmaceutical and Biomedical Analysis, 35, 633–638. DOI: 10.1016/j.jpba.2004.01.018. Polášek, M., Pospíšilová, M., & Urbánek, M. (2000). Capillary isotachophoretic determination of flufenamic, mefenamic, niflumic and tolfenamic acid in pharmaceuticals. Journal of Pharmaceutical and Biomedical Analysis, 23, 135–142. DOI: 10.1016/S0731-7085(00)00283-1. Pospíšilová, M., Polášek, M., & Jokl, V. (1998). Separation and determination of sorbitol and xylitol in multi-component pharmaceutical formulations by capillary isotachophoresis. Journal of Pharmaceutical and Biomedical Analysis, 17, 387–392. DOI: 10.1016/S0731-7085(98)00046-6. Pospíšilová, M., Polášek, M., & Procházka, J. (1997). Separation and determination of pharmaceutically important polyols in dosage forms by capillary isotachophoresis. Journal of Chromatography A, 772, 277–282. DOI: 10.1016/S00219673(96)00862-X. Posyniak, A., Zmudzki, J., & Niedzielska, J. (2001). Sample preparation for residue determination of gentamicin and neomycin by liquid chromatography. Journal of Chromatography A, 914, 59–66. DOI: 10.1016/S0021-9673(00)00980-8. Sádecká, J., Čakrt, M., Hercegová, A., Polonský, J., & Skačáni, I. (2001). Determination of ibuprofen and naproxen in tablets. Journal of Pharmaceutical and Biomedical Analysis, 25, 881–891. DOI: 10.1016/S0731-7085(01)00374-0. Sádecká, J., Hercegová, A., & Polonský, J. (1999). Determination of fenoprofen in serum by capillary isotachophoresis. Journal of Chromatography B: Biomedical Sciences and Applications, 729, 11–17. DOI: 10.1016/S0378-4347(99)00108-5. Sádecká, J., & Polonský, J. (1996). Determination of some cardiovascular drugs in serum and urine by capillary isotachophoresis. Journal of Chromatography A, 735, 403–408. DOI: 10.1016/0021-9673(95)00722-9. Valášková, I., Balážová, J., & Havránek, E. (1995). Monitoring of lithium levels in human serum after therapy with lithium preparations by capillary isotachophoresis. Journal of Chromatography B: Biomedical Sciences and Applications, 674, 310–313. DOI: 10.1016/0378-4347(95)00326-6.