Spectrofluorimetric determination of trace heparin using lomefloxacin-terbium probe

Spectrochimica Acta Part A 63 (2006) 241–246

Spectrofluorimetric determination of trace heparin using lomefloxacin-terbium probe Wei Wei, Hongjian Wang ∗ , Chongqiu Jiang Department of Chemistry, Shandong Normal University, Jinan 250014, China Received 13 December 2004; received in revised form 3 March 2005; accepted 4 March 2005

Abstract A new spectrofluorimetric method was developed for determination of trace amount of heparin (Hep). Using lomefloxacin (LOM)-terbium ion (Tb3+ ) as a fluorescent probe, in the buffer solution of pH 8.70, Hep can remarkably enhance the fluorescence intensity of the LOMTb3+ complex at λ = 545 nm and the enhanced fluorescence intensity of Tb3+ ion is in proportion to the concentration of Hep. Optimum conditions for the determination of Hep were also investigated. The linear range for the determination of Hep was 0.6–2.0 ␮g/ml and the detection limit was 45.22 ng/ml. This method is simple, practical and relatively free of interference from coexisting substances and can be successfully applied to assess Hep in biological samples. By the Rosenthanl graphic method, the association constant of Hep with the probe is 4.56 × 104 l/mol and binding numbers is 18.2. Moreover, the enhancement mechanism of the fluorescence intensity in the LOM-Tb3+ system and the LOM-Tb3+ -Hep system have also been discussed. © 2005 Elsevier B.V. All rights reserved. Keywords: Heparin; Lomefloxacin; Terbium; Spectrofluorimetric

1. Introduction Heparin (Hep) is a natural anticoagulant with an average molecular weight of about 15,000. It consists of repeating disaccharide units of uronic/glucuronic acid and glucosamine residues. Owing to the dissociation of acid groups, the whole Hep molecule is negatively charged in aqueous solution and the average charge is −70 [1]. It is a parenteral drug with a very rapid onset of action due to its inhibition of clotting factors near the end of the coagulation cascade. It has been widely used in many clinical procedures for more than 60 years and now is still the first-choice to prevent thromboses and to cure urgent vein thrombus [2]. Hep and its derivatives have a variety of biological activities such as anticoagulant, antilipemic, antithrombotic, immunoregulatory, antiphlogistic and antianaphylactic activities, etc. [3]. So the Hep level in the patients’ blood needs to be carefully and accurately monitored during surgery and recovery. Now the methods ∗

Corresponding author. Tel.: +86 531 2615258; fax: +86 531 2615258. E-mail address: [email protected] (H. Wang).

1386-1425/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.saa.2005.03.001

of determination of Hep can be classified in biological and chemical methods. The application of biological methods is confined because it is greatly affected by biological individuals and can’t be easily mastered [4]. The chemical methods include flowing injection analysis [5], ion-channel sensors [6], resonance Rayleigh scattering spectra [7,8], capillary chromatography [9], high-performance liquid chromatography [10], surface plasmon resonance sensor analysis [11], rotating electrode potentiometry [12], piezoelectric quartz crystal sensor [13] membrane electrode via protamine titration [14], extracorporeal membrane oxygenation [15] and so on. All of the methods mentioned above have high fluorescence quantum yield, large strokes shift, narrow emission bonds, a large fluorescence lifetime and hence avoid potential background fluorescent emission interferences from the biological matrix [16]. But there was no report about spectrofluorimetric method for the determination of Hep using LOM-Tb3+ as a fluorescent probe. LOM which is one kind of antibacterial containing ␣-carbonyl carboxylic acid configuration is an ideal ligand for Tb3+ . In this work, we chose lomefloxacin as the ligand of Tb3+ and investigated the possi-

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bility of the enhancement of the Tb3+ fluorescence sensitized by it and using Hep as co-ligand. Experimental results show that the characteristic peak of Tb3+ at 545 nm can be greatly enhanced and the enhanced fluorescence intensity is proportional to the concentration of Hep. Therefore, a new method with high sensitivity and selectivity for the spectrofluorimetric determination of heparin is established. This method is simple, relatively free of interference from coexisting substances and can be successfully applied to the determination of Hep sodium injection samples with satisfactory results. By the Rosenthal graphic method, the binding number and association constant of Hep with the probe were obtained. The mechanism of fluorescence enhancement between Tb3+ LOM complexes and LOM-Tb3+ -Hep was also studied.

2. Materials and methods 2.1. Apparatus All fluorescence measurements were carried out on an RF540 recording spectrofluorimeter (Shimadzu, Kyoto, Japan). A UV-265 recording spectrophotometer (Shimadzu, Kyoto, Japan) was used for UV spectra scanning and the determination experiments. All pH measurements were made with a pHs-3C digital pH meter (Shanghai Leici Device works, China).

buffer solution at pH 4.2. A small column (diameter = 1.2 cm) was made with the prepared CMC supported over quartz wool in an all-glass column (height 30 cm). The CMC is poured into the column to a height of 20 cm for the determination of Hep in blood serum. The prepared CMC is poured into another column (diameter = 1.2 cm, height = 60 cm) to a height of 50 cm for the determination of Hep in whole blood. Sephdex G-25 should be suspended in water for at least 6 h and then poured into the column (diameter = 1.2 cm, height = 30 cm) to a height of 20 cm. 2.3. General procedure To 10 ml color comparison tubes, solutions were added in the following order: 1.5 ml 5.0 × 10−4 mol/l Tb3+ ion solution, 1.5 ml 1.2546 × 10−5 mol/l LOM solution, 2.0 ml 10.0 ␮g/ml Hep solution, 1.0 ml buffer solution. The mixture was diluted to the mark with water and stand for 20 min at room temperature. The fluorescence intensity was measured at λex /λem = 335 nm/545 nm. The enhanced fluorescence intensity of LOM-Tb3+ by Hep was represented as F = F − F0 Here F and F0 are the fluorescence intensities of the systems with and without Hep, respectively.

2.2. Materials

3. Results and discussion

All chemicals used were of analytical-reagent or higher grade. Doubly distilled demineralized water was used for the preparation of all solutions and for all determinations. A stock Hep (Shanghai Chemical Reagent Company, China) solution (1.0 mg/ml) was directly dissolved in water. The working standard solution (10.0 ␮g/ml) was freshly prepared by appropriate dilution with water. A stock LOM (Biological Product Institution of Chinese Medicine) solution was directly dissolved in water. The working standard solution (1.2546 × 10−5 mol/l) was freshly prepared by appropriate dilution with water. All stocking solutions and working solutions given above were stored at 0∼4 ◦ C. A Tb3+ ion stock solution was prepared by dissolving Tb4 O7 (Shanghai Yuelong Chemical Plant, China) with a small amount of hydrochloric acid, then diluting to mark with hydrochloric acid (0.1 mol/l). The working solution (5.0 × 10−4 mol/l) was obtained by appropriate dilution of the stocking solution with water. A Tris–HCl buffer solution (0.05 mol/l, pH 8.70) was used for the system. A CMC column was prepared as follows: CMC was washed successively in 0.5 mol/l sodium hydroxide, 0.5 mol/l hydrochloric acid and 0.5 mol/l sodium hydroxide. The CMC is then suspended in 0.2 mol/l sodium acetate–acetic acid

3.1. Characteristics of fluorescence spectra The fluorescence excitation spectrum and emission spectrum of Tb3+ , LOM-Tb3+ , Tb3+ -Hep, LOM-Tb3+ -Hep, are shown in Fig. 1. It can be seen from Fig. 1 that single Tb3+ ion solution and Hep solution have nearly no peak. Comparing curve 1 with curve 4 in Fig. 1, after the addition of LOM into the Tb3+ ion solution, LOM can form a binary complex with Tb3+ ion. So it appears two little characteristic peaks of Tb3+ ion at 590 nm and 545 nm, and it is the 5 D0 –7 F1 transition and 5 D0 –7 F2 transition of Tb3+ ion, respectively. Comparing curve 4 with curve 7 in Fig. 1, it can be seen that the characteristic peak of Tb3+ at 545 nm can be enhanced remarkably after the addition of Hep, which indicates that Hep can form a very stable ternary complex with the LOM-Tb3+ system. 3.2. Effect of experimental conditions 3.2.1. Effect of pH The pH of the medium had great effect on the fluorescence intensity of the system, as shown in Fig. 2. The experimental results showed that the F reached maximum and remained constant at pH 8.40–9.00. Therefore pH 8.70 was selected with the using of 0.05 mol/l Tris–HCl buffer solution for further study. As the volume of buffer solution added 1.0 ml F

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Fig. 1. (a) Fluorescence excitation spectrum; (b) fluorescence emission spectrum. (1) Tb3+ , (2) lomefloxacin (3) Hep, (4) Tb3+ -lomefloxacin, (5) lomefloxacinHep, (6) Tb3+ -Hep, and (7) Tb3+ -lomefloxacin-Hep. Experimental conditions: LOM: 1.8819×10−6 mol/l, Tb3+ : 7.5×10−5 mol/l, Hep: 2.0 ␮g/ml, buffer: pH 8.70, λex /λem = 335 nm/545 nm.

and the concentration of LOM solution is 1.5055 × 10−6 to 2.5092 × 10−6 mol/l, Thus 1.8819 × 10−6 mol/l LOM (the added amount of LOM is 1.5 ml) was selected for further study. The composition ratio for the LOM to Tb3+ in the LOM-Tb3+ -Hep system is 1:39.85.

Fig. 2. Effect of pH.

reached maximum, then 1.0 ml was chosen in the following experiments. 3.2.2. Effect of time and the addition order of reagents Completing the chelation reaction of the LOM-Tb3+ -Hep system at room temperature needed 15 min at least. The fluorescence intensity then remained constant for 15–40 min. Therefore all measurements were made at 20 min for further study. Adding various reagents in different order had influence on the F, F0 and F. The experimental results indicate that it was optimum when solutions were added in the following order: Tb3+ , LOM, Hep and buffer. So this order was chosen in the following experiments. 3.2.3. Effect of the amount of LOM and Tb3+ The amount of LOM had the influence on the fluorescence intensities of the solutions. The experimental results showed that the F reached maximum and remained constant when the concentration of Tb3+ ion solution is 7.5 × 10−6 mol/l

3.2.4. Effect of coexisting substances Under the optimum conditions, a systematic study of various nonprotein substances in the determination of Hep (2.0 ␮g/ml) was carried out. The criterion for interference was fixed at a ±10% variation of the average fluorescence intensity calculated for the established level of Hep, the experimental results were Mo6+ 1.04 × 10−6 mol/l, Mn2+ 6.25 × 10−7 mol/l, Cu2+ 1.25 × 10−6 mol/l, Ca2+ 2.50 × 10−6 mol/l, Mg2+ 5.00 × 10−6 mol/l, Cd2+ 1.80 × 10−6 mol/l, thymine 1.85 × 10−6 mol/l, l-histidine 9.87 × 10−5 mol/l glutamic acid 2.00 × 10−4 mol/l, l-leucine 1.02 × 10−4 mol/l, saccharose 1.00 × 10−3 mol/l, lysozyme 5.00 × 10−6 mol/l.

4. Analytical application 4.1. Linear range and limit of detection Under the experimental conditions, there is a linear relationship between fluorescence intensity and Hep concentration in the range of 0.6–2.0 ␮g/ml with a correlation coefficient of 0.99832. The limit of detection was determined to be 45.22 ng/ml when the standard deviations was 0.1492 obtained from a series of 13 reagent blanks. By comparison with some existing methods, as shown in Table 1, the present methods have the advantages in the following terms: high sensitivity, good stability and wide linear range. It avoids potential background fluorescent emission interferences from the biological background. So this method may provide a new kind of luminescent probe for the

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Table 1 Comparison of spectrofluorometric methods for the determination of Hep Method

Linear range

Detection limit

References

Flowing injection analysis Ion-channel sensors Resonance Rayleigh scattering Capillary chromatography High-performance liquid chromatography Surface plasmon resonance sensor analysis Rotating electrode potentiometry Piezoelectric quartz crystal sensor This method

0–12 ␮g/ml 0.05–1.5 ␮g/ml 0–0.4 ␮g/ml 80–7000 U/la 0.002–5 pmol 0.2–2 U/mlc 0.05–0.5 U/mle 0–3 U/mlf 0.6–2.0 ␮g/ml

300 ng/ml 26 ng/ml 3.35 ng/ml 25 U/lb

[5] [6] [7,8] [9] [10] [11] [12] [13]

a b c d e f

0.2 U/mld

45.22 ng/ml

80–7000 U/l is equivalent to 0.504–44.1 ␮g/ml. 0.2–2 U/ml is equivalent to 1.26–12.6 ␮g/ml. 25 U/l is equivalent to 157.5 ng/ml. 0.2 U/ml is equivalent to 1260 ng/ml. 0.05–0.5 U/ml is equivalent to 0.315–3.15 ␮g/ml. 0–3 U/ml is equivalent to 0–18.9 ␮g/ml.

determination of biomolecular systems and can be applied to time-resolved fluoroimmunoassay. 4.2. Determination of heparin in injection The developed method was applied to the determination of Hep in injection. The results were shown in Table 2. For the assay of Hep, the samples must be diluted appropriately within the linear range of the determination of Hep and the sample solution was analyzed by the method developed above, using the standard calibration method. From Table 2, it can be seen that the developed method can be easily performed and affords good precision and accuracy when applied to real samples. 4.3. Determination of heparin in blood samples The developed method was applied to the determination of Hep in blood samples. Human serum albumin, hemoglobin and other proteins interfere seriously for the system. For the assay of Hep in blood samples, the samples can be applied to a carboxymethyl cellulose (CMC) column and a sephadex G-25 column. As the eluent for the CMC column, 0.2 mol/l sodium acetate–acetic acid buffer solution at pH 4.2 was chosen. Human serum albumin, hemoglobin and other proteins

are positive ions and Hep are negative ions when 0.2 mol/l sodium acetate–acetic acid buffer solution is at pH 4.2. The CMC column is a cation exchange column, so human serum albumin, hemoglobin and other proteins can be exchanged through the CMC column, whereas Hep can flow down and then directly into the sephdex G-25 column. The sephdex G25 column have a salting-out effect here in order to get rid of small molecule impurities. As a result, the interference of human serum albumin, hemoglobin and other proteins can be effectively removed. To 1.0 ml blood serum (comes from a normal person) mixed with 1.0 ml 10 mg/ml Hep. The mixture is applied to the CMC column followed by 60 ml 0.2 mol/l sodium acetate–acetic acid buffer solution at pH 4.2. The eluent is followed directly in the sephdex G-25 column and then eluted by 60 ml water. All of the eluent is collected into a 250 ml calibrated flask and then diluted. A volume of 1.0 ml of the worked sample solution, in which the concentration of Hep is 10 ␮g/ml, is analyzed by the method developed above. The whole blood was from a cardiac surgery patient in QiLu Hospital (Jinan, China). A volume of 1.0 ml whole blood sample (with 2.0 mg Hep in it) is applied to the CMC column followed by 150 ml 0.2 mol/l sodium acetate–acetic acid buffer solution at pH 4.2. The eluent (colorless) is directly applied to the sephdex G-25 column and then eluted

Table 2 Determination of Hep in samples (the standard value of the sample is 12,500 IU/2 ml) Sample no.

Sample added (␮g ml−1 )

Single found (␮g ml−1 )

Average (␮g ml−1 )

Conversion of mark value (IU/2 ml)

Average recovery (%)

R.S.D. (%)

20030310 a 020911-1b 0311111c 020504d

2.344 2.344 1.875 1.875

2.169, 2.322, 2.251, 2.287, 2.313 2.275, 2.307, 2.254, 2.269, 2.301 1.973,1.892, 1.909,2.006, 1.969 1.945, 1.872, 1.925, 1.977, 1.957

2.268 2.281 1.949 1.937

12100 12163 12988 12913

96.8 97.3 103.9 103.3

2.7 1.0 2.4 2.1

a b c d

Tianjin biochemical pharmaceutical factory of China. Maanshan plant of anhui xinli pharmaceutical Co. Ltd. of China. Wanbang biochemical pharmaceutical Co. Ltd. of China. Nanjing biochemical pharmaceutical Co. Ltd. of China.

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Table 3 Determination of Hep in blood samples Samples

Actual value (␮g/ml)

Measure value (␮g/ml)

Average (␮g/ml)

Average recovery (%)

R.S.D. (%)

Hep (via column) Blood serum mixed with Hepa Whole bloodb

2.00 2.00 2.00

2.012, 2.020, 2.025 2.083, 2.028, 2.042 2.054, 2.0922.039

2.019 2.051 2.061

101.0 102.6 103.1

0.3 1.4 1.3

a b

The blood serum was from a normal person. The whole blood was from a cardiac surgery patient in QiLu Hospital (Jinan, China).

by 60 ml water. All of the eluent is collected into a 250 ml calibrated flask. A volume of 1.25 ml of the worked sample solution, in which the concentration of Hep is 8 ␮g/ml, is analyzed by the method developed above. The result is shown in Table 3. From Table 3 it can be seen that the developed method can be easily performed and affords good precision and accuracy when applied to blood samples. 4.4. Measurement of association constant and binding numbers The Rosethanl graphic method [18], regarded as a modification of the ScaLOMhard method, was used to estimate the association constant (K) and the binding number (N) of the biomacromolecule to the LOM-Tb3+ probe. Briefly, when C, as a constant, is the biomacromolecule concentration in the system, and Cb , Cf and CTb 3+ -LOM are correspondingly the Hep-bound, free and total concentrations of the complex, the Rosenthanl plot follows Eq. (1) [19,20]. Cb = −(CTb3+ -LOM − Cf )K + NCK Cf

(1)

Since CTb 3+ -LOM = Cb + Cf , thus C = −(CTb3+ -LOM − Cf )K + NCK + 1 Cf

(2)

in the system, if CTb 3+ -LOM and Cf are within the dynamic range of the calibration graph for LOM-Tb3+ -Hep complex, Eq. (3) can be obtained. 
F0 F CTb3+ -LOM K + NCK + 1 (3) =− 1− F F0 F, F0 are the intensities of the systems with and without Hep. The plot of F0 /F versus [1 − (F/F0 )CTb 3+ -LOM ] can

be obtained. The values of K and N are 4.46×104 l/mol and 16.2. 4.5. Reaction mechanism LOM is one kind of antibacterial bacteriophages containing ␣-carbonyl carboxylic acid configuration. LOM is ideal ligand for Tb3+ ion and it can possibly sensitize the fluorescence intensity of Tb3+ ion via intramolecular energy transfer [17]. The coordination number of Tb3+ ion is 6–8. According to the experimental results of the mole ratio for LOM to Tb3+ given above, we can see that the coordination of Tb3+ ion is unsaturated. There are still a lot of positive charges and blank orbits in the LOM-Tb3+ complex. Hep, as a kind of biomacromolecule with an average molecular mass 15,000 and an average charge −70, is a polymer consisting of repeating tetrasaccharide unit. The structure of tetrasaccharide unit is shown in Fig. 3. Hep has three O-sulfate groups, two N-sulfate groups, and two carboxyl groups per tetrasaccharide unit. The O-sulfate and N-sulfate groups completely dissociate under the experimental conditions. The carboxyl group is weakly acidic, and the pKa of d-glucuronic acid in Hep is 3.6. So carboxyl groups can completely dissociate under the experimental conditions. Therefore, the whole Hep molecule exists as a big polyvalent anionic state in water solution. Because Hep has seven binding sites (five sulfate groups and two carboxyl groups) per tetrasaccharide unit and the Hep used here contains 42 monosaccharide units, the total binding number is 73.5 (42 × 7/4) per Hep molecule [3]. Therefore, a very stable ternary complex in close proximity with big degree of molecular conjugation and rigid structure can be formed by the electrostatic interactions and coordinations between Hep and LOM-Tb3+ binary complex.

Fig. 3. Structure of the tetrasaccharide unit of hep.

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