Bromide

Bromide

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Bromide Application

Determination in plasma and serum

Analytical principle

Photometry

Completed in

November 2001

Summary Bromide can be assayed in plasma or serum with the help of the procedure described here. Background levels in the general population can be measured as well as elevated bromide levels in plasma due to exposure at work or from environmental sources, such as fumigants containing bromide, inhalation narcotics or medication. After the proteins have been precipitated out of the plasma using trichloroacetic acid, phosphate buffer at a pH of 5.5 is added to an aliquot of the supernatant. A solution of the indicator phenol red (kmax = 356 nm) is also added. The bromide present in the solution is oxidised to bromine by the addition of chloramine T to the sample, thus causing the reaction shown below with phenol red being converted to bromophenol blue (kmax = 589 nm).

The absorption of the sample is subsequently measured with respect to a photometric reference solution at a wavelength of 589 nm using a double-beam photometer. Human plasma samples are spiked with known bromide concentrations and treated in the same manner as the samples to be tested in order to plot a calibration curve.

The MAK-Collection Part IV: Biomonitoring Methods, Vol. 10. DFG, Deutsche Forschungsgemeinschaft Copyright © 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 3-527-31137-8

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Bromide Within-series imprecision: Standard deviation (rel.) sw = 13.4% or 4.8% Prognostic range u = 34.3% or 12.3% at a concentration of 4 mg or 8 mg per litre plasma and where n = 6 determinations Between-day imprecision: Standard deviation (rel.) sw = 20.2% or 8.7% Prognostic range u = 43.1% or 18.4% at a concentration of 4 mg or 8 mg per litre plasma and where n = 16 determinations Accuracy:

Recovery rate or

r = 90% at 10 mg/L r = 85 to 96% (interlaboratory comparison)

Detection limit:

1 mg bromide per litre plasma

Bromide Bromide is taken into the human body naturally with food and drinking water. Values between 0.1 and 0.3 mg bromide per kg of body weight, i.e. approximately 8 mg/day, are given as the daily intake for a healthy adult [1]. Bromide has a long half-life of 12 to 14 days in human blood [2, 3]. It is mainly eliminated by the kidneys at an excretion rate of approximately 4 mg/day [4]. Like the chloride ion, the bromide ion is mainly distributed in the extracellular space [5]. Thus all body fluids, such as whole blood, serum/plasma and urine, contain bromide concentrations ranging from 3 to 12 mg/L with a mean value of approx. 5 mg/L [6]. Investigations on the background levels of bromide in whole blood carried out by Olszowy et al. on 183 randomly selected blood donors in Australia showed a mean value of 5.3 ± 1.4 mg bromide per litre blood. Our own investigations of the plasma of 74 healthy Central Europeans living in Germany resulted in a median of 4.77 mg bromide per litre plasma as the background level [7]. While these values are in good agreement, median values of 12.61 mg bromide per litre plasma were found in Chilean groups (blood donors from an urban population) and 11.06 mg bromide per litre plasma (farm workers who were not exposed to bromide compounds at work) [7]. This leads to the conclusion that no uniform international reference value can be given. The background levels, which may depend on the local eating habits of the population, must be determined in each case. In addition to the natural intake of bromide with food and drinking water, it has been taken, e.g. in the form of potassium bromide, since 1857 as a drug to treat epilepsy, in particular in the case of grand mal seizures that are resistant to therapy. The dosage is gradually increased; due to large inter-individual fluctuations the serum bromide levels are constantly checked during therapy. Therapeutic effects are shown from a serum level of 0.6 g bromide/L, and the first side-effects, such as vigilance deficiency, are to be expected for serum concentrations greater than 1 g/L [8]. Biomonitoring Methods, Vol. 10.

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Further medicinal products containing bromide are dextromethorphan hydrobromide, a constituent of many cough medicines (e.g. Wick MediNait®, Ratiopharm® cough medicine, Rhinotussal® capsules), and pyridostigmine bromide (Kalymin®, Mestinon®) that is also used as a prophylactic against inhibition of acetylcholinesterase [10]. Bromoureides, such as carbromal and bromisoval, were widely used in the 1970s, initially as a soporific available from pharmacies, but they were chronically misused and were repeatedly used for suicide attempts. The metabolic release of bromide from the molecule was suitable for detecting chronic abuse and acute intoxication. Rapid tests were developed for this purpose as well as other detection techniques [11]. Abuse dropped distinctly when the substances were made available only on prescription; both drugs have now become obsolete in Germany [9]. Halothane is an inhalation narcotic containing bromine that was widely used in past decades and is still in use. Its oxidative metabolism mediated by cytochrome P 450 yields trifluoroacetyl metabolites that are reported to be responsible for liver necroses. A milder form of liver toxicity is attributed to the release of free radicals and subsequent lipid peroxidation by the anaerobic P 450 biotransformation of halothane. Bromide is formed as a quantitatively significant metabolite in both metabolic pathways. It can be determined as a sum parameter for the P 450-mediated metabolism in a patient’s plasma. The difference between the pre-operative and post-operative bromide level (D Br–) reflects the halothane degradation and can serve as a measure of the release of potentially toxic metabolites. The difference between the pre-operative and post-operative bromide level (D Br–) was determined in a group of 24 patients. The mean value of the D Br– values was calculated as 32.7 ± 12 mg bromide per litre plasma, the median was 30.9 mg bromide per litre plasma [12]. The halogen alkanes methyl bromide, ethyl bromide and dibromoethane are important working materials containing bromide. While the use of the fungicide and antiknock agent dibromoethane has diminished considerably, ethyl bromide and especially methyl bromide are used as fumigants for the production, processing, storage and transport of food. This fumigation is stipulated for export to various countries (e.g. USA). One significant application is for disinfection of agricultural soil. After exposure, methyl bromide is converted in the human body to bromide and a methyl cation, which can react with cellular macromolecules. Therefore the measurement of plasma bromide levels is suitable for detecting inner levels after exposure to methyl bromide or ethyl bromide. Investigations carried out on a group of Chilean farm workers at the end of a fumigation season have shown that chronic exposure to methyl bromide is detectable compared with the background levels of the population. The median was found to be 15.4 mg bromide per litre plasma [7]. According to investigations by Verberk et al., neurotoxic effects of relevance to occupational medicine can be expected to occur from a bromide concentration of 20 mg per litre plasma or serum [13]. The plasma bromide level also serves as a significant laboratory parameter to measure the extent to which the starting compound has been metabolised in the case of intoxication with methyl bromide [14]. The Deutsche Forschungsgemeinschaft’s Commission for the Investigation of Health Hazards of Chemical Compounds in the Work Area has assigned methyl bromide to group 3 B of the carcinogenic working materials. In addition, the working group who Biomonitoring Methods, Vol. 10.

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assigns BAT values have stipulated a biological guideline value for methyl bromide of 12 mg bromide per litre plasma or serum [15]. This method can be used to monitor compliance with this biological guideline value. Author: M. Müller Examiners: W. Butte, T. Göen, M. Blaszkewicz

Biomonitoring Methods, Vol. 10.

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Bromide Application

Determination in plasma and serum

Analytical principle

Photometry

Completed in

November 2001

Contents 1 2 2.1 2.2 2.3 2.4 3 3.1 3.2 4 4.1 5 6 7 8 9 9.1 9.2 9.3 9.4 10 11

General principles Equipment, chemicals and solutions Equipment Chemicals Solutions Calibration standards Specimen collection and sample preparation Specimen collection Sample preparation Operational parameters Photometric working conditions Analytical determination Calibration Calculation of the analytical result Standardisation and quality control Evaluation of the method Precision Accuracy Detection limit Sources of error Discussion of the method References

1 General principles After the proteins have been precipitated out of the plasma using trichloroacetic acid, phosphate buffer at a pH of 5.5 is added to an aliquot of the supernatant. A solution of the indicator phenol red (kmax = 356 nm) is also added. The bromide present in the solution is oxidised to bromine by the addition of chloramine T to the sample, thus The MAK-Collection Part IV: Biomonitoring Methods, Vol. 10. DFG, Deutsche Forschungsgemeinschaft Copyright © 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 3-527-31137-8

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causing the reaction shown below with phenol red being converted to bromophenol blue (kmax = 589 nm).

The absorption of the sample is subsequently measured with respect to a photometric reference solution at a wavelength of 589 nm using a double-beam photometer. Pooled human plasma is spiked with known bromide concentrations and treated in the same manner as the samples to be tested in order to plot a calibration curve.

2 Equipment, chemicals and solutions 2.1 Equipment Double-beam spectrophotometer (e.g. Kontron Uvikon 922) Half-microquartz cells (1 cm layer thickness) with cover (e.g. from Hellma) Alternatively: Plastic cells (1 cm layer thickness) with cover (e.g. from Sarstedt) pH meter with a single-rod electrode (e.g. from WTW) Centrifuge (e.g. from Beckman) Magnetic stirrer (e.g. from Heidolph) Test-tube shaker (e.g. Vortex) Stopwatch that displays seconds (e.g. from TCM) Drying cupboard (e.g. from Heraeus) Volumetric flasks, 10 and 100 mL (e.g. from Brand) 250 mL Glass beakers (e.g. from Brand) Finn pipette 1 to 5 mL


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Microlitre pipettes, variable between 10 and 100 lL (e.g. from Eppendorf) Microlitre pipettes, variable between 100 and 1000 lL (e.g. from Eppendorf) Disposable syringes with or without an anticoagulant (e.g. Monovettes with or without K-EDTA from Sarstedt) 10 mL Polyethylene tubes 2 mL Polyethylene sample vessels, sealable (e.g. from Eppendorf)

2.2 Chemicals Phenol red (phenolsulphonephthalein) (e.g. Merck, No. 159375) Chloramine T (N-chloro-4-toluenesulphoamide, sodium salt) p.a. (e.g. Merck, No. 102426) Trichloroacetic acid, p.a. (e.g. Merck, No 100807) Sodium bromide, p.a. (e.g. Merck, No 106360) Potassium hydrogen phosphate, p.a. (e.g. Merck, No 104877) Disodium hydrogen phosphate dihydrate p.a. (e.g. Merck, No. 106580) Sodium thiosulphate, anhydrous (e.g. Merck, No 106512) Sodium hydroxide, p.a. (e.g. Merck, No 106495) Deionised water (e.g. produced by means of Millipore® technology)

2.3 Solutions 30% Trichloroacetic acid: 30 g trichloroacetic acid are weighed in a 100 mL volumetric flask and dissolved in bidistilled water. The flask is subsequently filled to its nominal volume with water. This solution can be stored at room temperature for approximately 4 weeks. Phosphate buffer pH 5.5: 0.2 g disodium hydrogen phosphate dihydrate and 3 g potassium dihydrogen phosphate are weighed in a 250 mL glass beaker and then dissolved in approx. 70 mL bidistilled water. The solution is transferred to a 100 mL volumetric flask, the beaker is rinsed with bidistilled water and the rinsing water is added to the flask. Then the flask is filled to its nominal volume with bidistilled water. The pH value of the solution is checked using a pH meter and should be 5.5 (± 0.1). This solution can be stored at room temperature for approximately 4 weeks.


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1 M NaOH: Approximately 40 mL bidistilled water are placed in a 250 mL glass beaker. A total of 4 g sodium hydroxide is added and dissolved by stirring with a magnetic stirrer. After being cooled with bidistilled water, the solution is transferred to a 100 mL volumetric flask, which is filled to a total volume of 100 mL. This solution can be stored at room temperature for approximately 4 weeks. Phenol red stock solution: Approx. 25 mg phenol red (phenolsulphonephthalein) are weighed in a 100 mL volumetric flask and dissolved in 5 mL of the 1 M NaOH solution. The flask is filled to its nominal volume with bidistilled water. The resulting stock solution must be freshly prepared before each measurement. Phenol red reagent solution: 5 mL of the phenol red stock solution are pipetted into a 10 mL volumetric flask. The flask is then filled to its nominal volume with 1 M NaOH solution. The resulting reagent solution must be freshly prepared before each measurement. 0.5% Chloramine T solution: 50 mg chloramine T (N-chloro-4-toluenesulphamide, sodium salt) are weighed in a 10 mL volumetric flask and dissolved in approx. 5 mL bidistilled water. The flask is subsequently filled to its nominal volume with bidistilled water. This solution must be freshly prepared before each measurement. 2.5% Sodium thiosulphate solution: 250 mg anhydrous sodium thiosulphate are weighed into a 10 mL volumetric flask and dissolved in approx. 5 mL bidistilled water. The flask is subsequently filled to its nominal volume with bidistilled water. This solution must be freshly prepared before each measurement.

2.4 Calibration standards Starting solution: Approximately 51.4 mg sodium bromide are weighed exactly in a 10 mL volumetric flask. The flask is filled to its nominal volume with bidistilled water (c = 4 g bromide/L). This solution can be stored at room temperature for approximately 4 weeks. Stock solution: Approx. 5 mL bidistilled water are placed in a 10 mL volumetric flask. 2.5 mL of the starting solution are added with a pipette. The flask is subsequently filled to its nominal volume with bidistilled water (c = 1 g bromide/L). This solution can be stored at room temperature for approximately 4 weeks.


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Calibration standards (8 to 128 mg/L) Calibration standards in bidistilled water are prepared from the stock solution in accordance with the following pipetting scheme. For this purpose the volumes of the stock solution shown in Table 1 are each filled into a 10 mL volumetric flask using a pipette, and then the flask is filled to its nominal volume with water. The resulting calibration standards must be freshly prepared before each measurement. Table 1. Pipetting scheme for the preparation of the aqueous calibration standards Volume of the stock solution [lL]

Final volume of the calibration standard [mL]

Concentration of the calibration standard [mg/L]

80 160 320 640 1280

10 10 10 10 10

8 16 32 64 128

Calibration standards in pooled human plasma are prepared from these aqueous calibration standards as described in Section 3.2.

3 Specimen collection and sample preparation 3.1 Specimen collection In order to determine the background levels of bromide as part of investigations in occupational medicine and environmental medicine, the person to be tested may not have been exposed to bromide, bromine or working materials or medication containing bromide for 60 to 70 days (equivalent to 5 times the half-life of the bromide ion [2, 3]) prior to testing. The inevitable intake of bromide with food and drinking water is negligible. Blood can be withdrawn at any time after this period of abstinence. The stipulated withdrawal time, which depends on the toxicokinetics of the individual substances, should be observed in order to establish occupational or environmental exposure to bromide itself or to bromide released from a xenobiotic. Disposable syringes with or without an anticoagulant (e.g. Monovettes with or without potassium EDTA from Sarstedt) are used to collect blood samples. The withdrawn blood is centrifuged at room temperature for 10 minutes at 1800 rpm (540 g). The supernatant is transferred to 10 mL polyethylene tubes and can be transported at room temperature, frozen at –80 8C or prepared for analysis. The samples frozen at –80 8C can be stored for at least 6 months without losses. Biomonitoring Methods, Vol. 10.

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3.2 Sample preparation 400 lL plasma are pipetted into a 2 mL polyethylene sample vessel (e.g. from Eppendorf) and 100 lL bidistilled water (for plasma blank samples or patient samples) or 100 lL of the aqueous calibration standards (to plot a calibration curve) prepared as described in Section 2.4 are added in each case. 100 lL 30% trichloroacetic acid are added to precipitate proteins, the sample vessel is sealed and the sample is intensively mixed on the Vortex for 10 s. The sample is incubated overnight at 37 8C in the drying cupboard or in the water bath. After incubation, the sample is cooled in the refrigerator at 4 to 8 8C for at least 20 minutes and then centrifuged at room temperature for 30 minutes at 13,000 rpm (12,000 g).

4 Operational parameters 4.1 Photometric working conditions A wavelength of 589 nm is adjusted on the double-beam spectrophotometer or an appropriate filter is used. The sample is measured with respect to the given reference solution (see Section 5). The instructions of the manufacturer of the equipment should be observed.

5 Analytical determination The photometric reference solution is prepared by pipetting 100 lL of the precipitation supernatant of a plasma blank sample into a half microquartz cell, adding 50 lL of the phenol red reagent solution and 550 lL of the phosphate buffer pH 5.5 and mixing them. A yellow solution is obtained. At the same time 100 lL of the precipitation supernatant of a sample or of a plasma calibration standard prepared as described in Section 3.2 are pipetted into a new half microquartz cell, 50 lL of the phenol red reagent solution and 400 lL of the phosphate buffer pH 5.5 are added, and they are mixed. Finally, 75 lL of the 0.5% Chloramine T solution are added, the sample is sealed with the cover and shaken. The colour reaction is stopped after exactly one minute (stopwatch!) by the addition of 75 lL of the 2.5% sodium thiosulphate solution; for this purpose the sample is sealed with the cover and shaken. The absorption of the reddish-violet solution is measured with respect to the solution for photometric comparison at a wavelength of 589 nm and a layer thickness of 1 cm. The coloured substance formed in this reaction is stable for at least one hour under these conditions. Duplicates of both the plasma samples and the plasma calibration standards are to be prepared and analysed in the manner described here. The mean value of the meaBiomonitoring Methods, Vol. 10.

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sured absorption is calculated in each case. A quality control sample is analysed with each analytical series (see Section 8). If the concentration of bromide in the sample to be analysed is too high due to poisoning or the intake of medication, the plasma can be diluted as described in Section 9.2.

6 Calibration Two plasma blank samples and 5 plasma calibration standards prepared as described in Section 3.2 are required to plot a calibration curve. The blank samples and plasma calibration standards are processed in accordance with Section 3.2 and analysed in duplicate determinations as described in Sections 4 and 5. While the first plasma blank sample serves as a solution for photometric comparison, the second plasma blank value is treated in the same manner as a test sample. The mean absorption value measured in this case is equivalent to the bromide content of the unspiked pooled plasma. This mean absorption value is subtracted from the mean absorption values of the analysed plasma calibration standards. The mean absorption values thus obtained are plotted with respect to the bromide concentrations in plasma in mg/L. The bromide concentrations in plasma are obtained by dividing the concentrations of the spiked aqueous calibration standards (cf. Section 2.4) by a factor of 4 to reflect the dilution in plasma (as 4 parts of the solution are composed of plasma and only one of water). The calibration curve is linear for concentrations between 2 and 32 mg bromide per litre plasma measured at 589 nm and 1 cm layer thickness. Calibration standards should be freshly prepared in plasma and included in each analytical series to plot a completely new calibration curve. Figure 1 shows an example of a calibration curve in human plasma in the given range.

7 Calculation of the analytical result The mean absorption values obtained for the unknown content in the plasma samples are used to read off the corresponding concentration in mg bromide per litre plasma from the calibration graph.

8 Standardisation and quality control Quality control of the analytical results is carried out as stipulated in the guidelines of the Bundesärztekammer (German Medical Association) [16] and in the special preliminary remarks to this series. In order to check the precision, one plasma control sample containing a constant concentration of bromide is analysed in each analytical series. As material for quality control is not commercially available, it must be prepared in the laboratory. Biomonitoring Methods, Vol. 10.

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A six-month supply of the control material is prepared, divided into aliquots in sealable 2 mL polyethylene sample tubes and stored in the deep-freezer at –80 8C. Control material thus stored is stable for at least 6 months. The theoretical value and the tolerance range for this quality control material are determined in the course of a pre-analytical period (one analysis of the control material on each of 20 different days) [17, 18]. External quality control can be achieved by participation in round-robin experiments. However, to the best of our knowledge the round-robin experiments for occupational and environmental toxicological analysis carried out in Germany do not include bromide in their external quality control programme at present [19].

9 Evaluation of the method 9.1 Precision Pooled human plasma from persons who were not occupationally exposed to bromide were spiked with 4 or 8 mg bromide per litre plasma, processed and analysed to determine the precision in the series. Six replicate determinations of the plasma samples yielded the precision in the series documented in Table 2. In addition, the precision from day to day was determined. The same material was used as for the determination of the precision in the series. These plasma solutions were processed and analysed on 16 different days. The precision from day to day for bromide thus obtained is also given in Table 2. Table 2. Precision in the series and from day to day for the determination of bromide in plasma Spiked concentration [mg/L]

Standard deviation (rel.) Prognostic range [%] [%]

Precision in the series (n = 6) Plasma 4 8

13.4 4.8

34.3 12.3

Precision from day to day (n = 16) Plasma 4 8

20.2 8.7

43.1 18.4

9.2 Accuracy Recovery experiments were performed to check the accuracy of the method. Ten different human plasma samples were each spiked with 10 mg bromide per litre plasma and analysed for this purpose. Analysis of the unspiked and spiked plasma samples showed a mean recovery rate of 90%. In addition, the accuracy of the method was checked in an inter-laboratory comparison. For this purpose 5 serum samples of patients who were taking potassium broBiomonitoring Methods, Vol. 10.

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mide as treatment for epilepsy were investigated. The comparative laboratory determined its values using a gold chloride method that yields reliable data from a concentration of 75 mg bromide per litre serum and is regarded as a standard clinical method [8, 20]. The bromide levels in serum determined using this method were between 1.35 and 2.39 g bromide per litre serum. As the method described here is approximately 30 times more sensitive, aliquots of the prepared patient samples were diluted with the prepared plasma blank sample of pooled human plasma and then measured. The bromide content of the pooled human plasma was determined separately and subtracted from the determined bromide content of the diluted patient sample. In comparison to the gold chloride method, recovery rates of 85 to 96% of the bromide levels in the serum of the patients’ samples were calculated by extrapolation, showing a very good correlation between the two methods [21].

9.3 Detection limits Under the given conditions for sample preparation a detection limit of 1 mg bromide per litre plasma was estimated.

9.4 Sources of error The background level of bromide in plasma is mainly the result of the inevitable intake of the anion with food and drinking water [6]. The source of elevated bromide levels may be exposure to bromide or bromine at work or from the environment as well as working materials and medication containing bromine. In order to determine a specific exposure to bromide or a bromide-releasing extraneous substance, it is necessary to rule out exposure to bromide from another source by careful study of the occupational and environmental medical history of the person to be tested. It is important to take medication containing bromide into consideration, such as dextromethorphan hydrobromide, a constituent of many cough medicine (e.g. Wick MediNait®, Ratiopharm® cough medicine, Rhinotussal® capsules), and pyridostigmine bromide (Kalymin®, Mestinon®) that is also used as a prophylactic against inhibition of acetylcholinesterase [10]. As the photometric determination of bromide is based on its oxidation to bromine and the subsequent conversion with phenol red to the indicator bromophenol blue, it is necessary to comply with the given reaction conditions exactly in order to achieve valid and reproducible results. All the solutions used must be at room temperature. The concentrations of the reaction solutions must be exactly as specified and the pH value of the phosphate buffer must be adjusted correctly. The reaction time for the formation of bromophenol blue of one minute may not be shortened or exceeded. It must be ensured that the solutions are effectively and rapidly mixed, especially when the reaction is stopped. It is essential to take the stability data of the individual solutions given in Sections 2.3 and 2.4 into account. Biomonitoring Methods, Vol. 10.

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Plasma samples with bromide concentrations outside the calibration range cannot be simply diluted with water or buffer, then processed and analysed anew using this method. The amount of trichloroacetic acid used for precipitation is intended to precipitate a certain quantity of protein. Dilute samples contain less protein; as a consequence an excess of trichloroacetic acid remains in the precipitation supernatant and it cannot be buffered in the photometric reaction solution. As both phenol red and bromophenol blue are pH indicators, a yellowish solution is formed after the reaction under these acidic conditions. The resulting solution does not yield absorption values at 589 nm that can be evaluated. Inorganic constituents present in plasma do not cause interference to the bromide assay described here, and fluoride and chloride ions, in particular, behave neutrally. Iodide is an exception, it can interfere with the colour reaction on principle. However, free iodide is present in serum only in concentrations of 5 to 25 lg iodide per litre serum [22], therefore at a detection limit of 1 mg bromide per litre plasma for this method it is a negligible source of interference in practice. Interference to the reaction can be caused by excessively high bromide concentrations in plasma, as an excess of released bromine destroys the bromophenol blue that is formed and thus lower bromide concentrations in the sample are simulated [23]. Therefore measurement must always be carried out in the given linear calibration range for this reason.

10 Discussion of the method The method presented here is suitable for determination of the background level of bromide in plasma as well as elevated plasma bromide levels due to occupational or environmental exposure. Although the principle on which the measurement is based has been known for a long time [11, 20, 23], the method described here represents an important further development with regard to sensitivity, as the detection limit of the procedure is 1 mg bromide per litre plasma. Exact data on the precision, accuracy and sources of interference permit the establishment of a robust routine measurement procedure. Compared with other recognised methods for bromide assay, such as X-ray fluorescence analysis [6, 24], neutron activation analysis [25], ICP-MS [4] and ion chromatography [26], the method described here is remarkable, as it does not require apparatus of great technical complexity. Thus exact and valid bromide measurements can be performed, even in a simply equipped laboratory. One of the examiners of the method has pointed out that a plate photometer can be used if an increase in the sample throughput is desired. Instruments used: Kontron Uvikon 922 double-beam spectrophotometer


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11 References [1] M. Janghorbani, T.A. Davis and B.T. Ting: Measurement of stable isotopes of bromine in biological fluids with inductively coupled plasma mass spectrometry. Analyst 113, 405–411 (1988). [2] R. Söremark: The biological half-life of bromide ions in human blood. Acta physiol. scand. 50, 119–123 (1960). [3] N. Vaiseman, G. Koren and P. Pencharz: Pharmacokinetics of oral and intravenous bromide in normal volunteers. J. Toxicol. Clin. Toxicol. 24, 403–413 (1986). [4] P. Allain, Y. Mauras, C. Douge, L. Jaunault, T. Delaporte and C. Beaugrand: Determination of iodine and bromine in plasma and urine by inductively coupled plasma mass spectrometry. Analyst 115, 813–815 (1990). [5] A.G. Rauws: Pharmacokinetics of bromide ion – an overview. Food Chem. Toxicol. 21, 379– 382 (1983). [6] H.A. Olszowy, J. Rossiter, J. Hegarty and P. Geoghegan: Background levels of bromide in human blood. J. Anal. Toxicol. 22, 225–230 (1998). [7] M. Müller, X. Barraza, P. Reinhold, G. Westphal, J. Bünger, M. Zeise and E. Hallier: Bestimmung des Plasmabromidspiegels als arbeits- und umweltmedizinischer Belastungsparameter. Verh. Dt. Ges. Arbeitsmed. 41, 319 – 322 [8] H. E. Boenigk, J.H. Lorenz and U. Jürgens: Aktuelle Erfahrungen mit Bromiden zur Behandlung generalisierter Epilepsien. In: R. Kruse (ed.): Epilepsie 84, Einhorn-Presse Verlag, Reinbeck, 316–325 (1985). [9] Rote Liste® Service GmbH: Rote Liste 2000. Editio Cantor Verlag, Aulendorf (2000). [10] L. Szinicz: Chemische Kampfstoffe. In: H. Marquardt and S.G. Schäfer (eds.): Toxikologie. BIWissenschaftsverlag, Mannheim 571–588 (1994). [11] W. Butte and R. Wronski: A universally applicable rapid bromine test. Arch. Toxicol. 36, 147– 149 (1976). [12] M. Müller, B. Dreeßen, M. Hatting, J. Bünger and E. Hallier: Identification of the halothane metabolite N-acetyl-S-(2-chloro-1-fluorovinyl)-L-cysteine in human urine by HPLC-ESI-ion trap mass spectrometry. N-S Arch. Pharmacol. 361, No. 4 Suppl., R 144 (2000). [13] M.M. Verberk, T. Rooyakkers-Beemster, M. de Vlieger and A.G.M. van Vliet: Bromine in blood, EEG and transaminases in methyl bromide workers. Br. J. Ind. Med 36, 59–62 (1979). [14] R. Garnier, M.O. Rambourg-Schepens, A. Müller and E. Hallier: Glutathione transferase activity and formation of macromolecular adducts in two cases of acute methyl bromide poisoning. Occup. Environ. Med. 53, 211–215 (1996). [15] Deutsche Forschungsgemeinschaft: List of MAK and BAT values 2005, 41st report. Wiley-VCH, Weinheim (2005). [16] Bundesärztekammer. Richtlinie der Bundesärztekammer zur Qualitätssicherung quantitativer laboratoriumsmedizinischer Untersuchungen. Dt. Ärztebl. 100, A3335 – A3338 (2003). [17] J. Angerer and G. Lehnert: Anforderungen an arbeitsmedizinisch-toxikologische Analysen – Stand der Technik. Dt. Ärztebl. 37, C1753 – C1760 (1997). [18] J. Angerer, T. Göen and G. Lehnert: Mindestanforderungen an die Qualität von umweltmedizinisch-toxikologischen Analysen. Umweltmed. Forsch. Prax. 3, 307–312 (1998). [19] Ringversuch Nr. 34. Qualitätsmanagement in der Arbeits- und Umweltmedizin, Projektgruppe Qualitätssicherung, Organisation: Institut für Arbeits-, Sozial- und Umweltmedizin der Universität Erlangen-Nürnberg (2004). [20] B. Lange and Z.J. Vejdelek: Brom und Bromide: Photometrische Analyse. Verlag Chemie, Weinheim, 331–335 (1980). [21] M. Müller, P. Reinhold, M. Lange, M. Zeise, U. Jürgens and E. Hallier: Photometric determination of human serum bromide levels – a convenient biomonitoring parameter for methyl bromide exposure. Toxicol. Letters 107, 155–159 (1999). [22] S.H. Ingbar: Krankheiten der Schilddrüse. In: P.W. Straub (ed.) Harrison – Prinzipien der Inneren Medizin. Schwabe & Co. AG Verlag, Basel (1989).


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[23] W. Kisser: Über den Nachweis und die quantitative Bestimmung bromierter Harnstoffderivate in der Toxikologie. Arch. Toxikol. 22, 404–409 (1967). [24] I.G. Stump, J. Carruthers and J.M. D’Auria: Quantitative analysis of trace elements in human blood and plasma by energy dispersive X-ray fluorescence. Clin. Biochem. 10, 127–132 (1977). [25] M.S. Rapaport, M. Mantel and R. Nothmann: Determination of bromine in blood serum by neutron activation analysis and X-ray spectrometry. Anal. Chem. 51, 1356–1358 (1979). [26] Y. Nagamine, Y. Hamai, K. Chikamori, T. Kita, M. Hirohita, I. Oshima, S. Yamashita and K. Shima: Asymptomatic hyperbromidaemia detected as pseudohyperchloridaemia measured with an ion selective electrode meter. Scand. J. Clin. Lab. Invest. 48, 177–182 (1982).

Author: M. Müller Examiners: W. Butte, T. Göen, M. Blaszkewicz

Fig. 1. Calibration curve, prepared in human plasma
