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A rapid method for the emergency analysis of paraquat in plasma using second derivative spectroscopy
153
Clinica Chimica Actu, 117 (1981) 153-165 Elsevier/North-Holland Biomedical Press
CCA
1946
A rapid method for the emergency analysis of paraquat in plasma using second derivative spectroscopy D.R. Jar-vie a, A.F. Fell b,* and M.J. Stewart
’
a Toxicology
Unit, Department of C&al Chemistry, Royal Infirmary, Edinburgh EH3 9Y W, ’ Department of Pharmacy, Heriol- Watt University, Edinburgh EHI 2HJ and ’ Department of Clinical Biochemistry, Royal Infirmary, Glasgow G4 OSF (UK) (Received
April 16th. 1981)
A rapid method for the analysis of paraquat in plasma of poisoned patients is described. The dithionite colour reaction is used to detect paraquat, and matrix interference is eliminated by the use of a chemical deproteinization technique followed by second derivative spectroscopy of the reaction mixture. The method was compared with an established procedure using ion-pair extraction followed by dithionite reduction. Sensitivity and precision were comparable and the correlation in patients’ plasma was good. The proposed direct method is rapid and suitable for emergency use in the management of patients poisoned with .paraquat.
Introduction
The prognosis for the patient severely poisoned with paraquat is poor, unless the poison can be eliminated from the body before’ it causes irreversible damage to sensitive tissues. Techniques such as haemodialysis and haemoperfusion which remove paraquat from the plasma space must be instituted as early as possible, while the plasma concentration is high, if they are to have any likelihood of success [l]. Methods for the measurement of plasma paraquat concentration as a basis for treatment must therefore be both rapid and precise. Rapid radioimmunoassay methods are available [2-41, but this technique is not always suitable for use in the general clinical chemistry laboratory. Of the calorimetric assays based on dithionite reduction of paraquat, that of Knepil [5] is rapid but subject to interference from endogenous materials in plasma. It is therefore unsuitable for accurate determina* To whom correspondence OC09-8981/81
should be addressed.
/OOOC-0000/$02.75
0 1981 Elsevier/North-Holland
Biomedical
Press
154
tion at the lower levels, around 0.1 mg/l, .at which clinical decisions may be required [l]. Such interference is eliminated by ion-pair extraction in the method of Jarvie and Stewart [6,7], which has given good service in recent years for the care of poisoned patients in our respective hospitals. This method has adequate sensitivity, but requires good pipetting technique and takes about 40 min for the complete analysis of a specimen. Derivative spectroscopy (8- 1 l] offers an alternative approach to the reduction of background absorbance. In this mode the normal (zero-order) absorption spectrum is transformed to a function of its second (d2A/dA2) or higher even derivative, by an analog or digital device as the spectrum is scanned. Such modules are available as relatively inexpensive accessories for several well-known commercial spectrophotometers, so that the technique is well within the scope of the smaller laboratory. In derivative mode, sharp features such as the paraquat cation radical peak are enhanced, to give improved resolution of overlapping bands, whereas broader absorption bands such as those arising from non-specific matrix absorption are suppressed. Recent studies have characterized the behaviour of the second and fourth derivative spectra of the paraquat cation radical [lo]. Derivative detection confers four major advantages over the zero order mode. The interference due to diquat, often co-formulated in commercial weedkiller preparations, is almost eliminated in derivative mode and the need for baseline correction is therefore avoided. Slight differences in dithionite concentration between test and reference cuvettes seriously disturb the zero-order absorption spectrum, but have less effect in derivative mode, thus conferring increased precision at lower paraquat concentrations. The interference of endogenous substances is reduced, implying that extraction may be replaced by a simpler purification stage. Because of the enhanced sensitivity in the derivative mode, a concentration step is no longer required. The present work describes the development and evaluation of a rapid method of paraquat estimation in plasma by derivative spectroscopy after protein precipitation and dithionite reduction. The method has a detection-limit of 0.05 mg/l paraquat dichloride, is simple to perform and is suitable for emergency use. Materials and methods Instrumentation A model SP8-100 scanning spectrophotometer, equipped with two electronic derivative modules (Pye-Unicam Ltd., Cambridge, UK), was used with a matched pair of quartz semimicro cuvettes of. 1 cm path-length (Pye-Unicam Ltd). Simple modification to isolate the microswitch actuating an optical filter at 390 nm was necessary to eliminate the electrical interference spike on the derivative spectrum. Derivative spectroscopy The second and fourth derivative spectra of the absorption peak of the paraquat cation radical at 396 nm were generated using the optimized instrumental conditions summarized in the legend to Table II. The derivative amplitudes were measured
155
from peak to long-wavelength satellite (see Fig. 1); this measurement has been shown to be more sensitive and precise in paraquat analysis than measurement from peak to derivative baseline zero [lo]. The effect of random noise was reduced by recording each derivative spectrum three times and using the average derivative amplitude for analytical calculations. In the initial investigations of linearity and precision, both second and fourth derivative spectra were examined. Since the fourth derivative was found to give comparable results and conferred little advantage in terms of precision and sensitivity in the present work, second derivative measurements alone were used for the analysis of patients’ samples. Paraquat standard
Paraquat dichloride (Aldrich Chemicals, Poole, Dorset, UK) was dried to constant weight before use (2 h at 1OO’C) and subsequently stored in a desiccator. Stock solutions (20 and 200 mg/l) were prepared in distilled water, and serum standards made from them by dilution in horse serum (Horse Serum 3, Wellcome Reagents Ltd., Becker&am, Kent, UK).
Fourth
Second
Zero
I
A-
I
I
I
I
t
t
0.05A
I.021
+
1
I
DL
Lt
I
Ii
DL
i
Dz
I I
390
430
390
430
I
Dz
’ Is-
r t
390
430
h,nm Fig. 1. Zero order, second and fourth derivative spectra of 0.50 mg/l paraquat dichloride in serum after deproteinization with sulphosalicylic acid and reduction with alkaline dithionite. Instrumental conditions as in Table II (0.10-1.00 mg/l). The graphical measures available are: D,, peak to long wavelength satellite; D,, peak to derivative baseline zero. The paraquat cation radical peak is observed at 396 nm (zero order), 390 run (second derivative) and 387 nm (fourth derivative) due to the time constants of the electronic differentiators employed, which lead to a wavelength shift in the direction of scanning.
156
Reagents (1) Sulphosalicylic
acid, 100 g/l. A solution of 5-sulphosalicylic acid (Analar, B.D.H. Chemicals Ltd., Poole, Dorset, UK) in distilled water. (2) Alkaline dithionite solution, 20 g/l. 0.2 g sodium dithionite was dissolved in 10.0 ml aqueous 5 mol/l NaOH immediately before use in a batch of analyses. The reagent is stable for approximately 2 h.
Patients’ specimens All samples analysed for paraquat were taken from poisoned patients admitted during the previous 12 months to the Regional Poisoning Treatment Centre, Royal Infirmary of Edinburgh. Paraquat-free samples for the study of spectral interference were collected locally in the routine clinical chemistry laboratory. Blood was collected in tubes containing lithium heparin as anticoagulant. Plasma was separated on receipt in the laboratory, analysed for paraquat by the standard method [6] and then stored at -20°C pending repeat analysis by the protein precipitation method.
Analytical procedure To 1.0 ml serum or plasma in a lo-ml plastic tube add 1.0 ml 100 g/l sulphosalicylic acid solution. Mix thoroughly (10 s) by vortex mixer and centrifuge at 1500 X g for 5 min in a bench centrifuge. Decant the supernatant (total vol. 1.6 ml). To 1.0 ml of the supernatant in a second plastic tube add 0.25 ml alkaline dithionite solution. Mix thoroughly and transfer to a 1.0 cm seminicro quartz cuvette. Record the second derivative spectrum using the conditions described in Table II and scanning from 430 to 370 nm, using as reference a fresh mixture of 1.0 ml distilled water and 0.25 ml alkaline dithionite solution. Process a paraquat dichloride standard (1 .OO mg/l in horse serum) in the same manner as the test samples and analyse before and after each batch of tests. Measure the derivative amplitude in tests and standards from the peak minimum at 390 nm to the long-wavelength satellite maximum at 396 nm (mean of 3 scans). If haemolysis is present, measure to the precise wavelength of the long-wavelength satellite, as recorded in the spectrum of the 1.00 mg/l paraquat standard. The paraquat dichloride concentration in the test samples is calculated by linear proportion. Results
(I) Development of the analytical method Choice of protein precipitant Five standard techniques of protein precipitation were applied to horse serum (Table I). The technique specifically designed for paraquat determination by Knepil [5] was also tested. Of these six techniques, only sulphosalicylic acid proved reliable enough to form the basis of an analytical method. Similar results were obtained using pooled human plasma. A final sulphosalicylic acid concentration of 25 g/l was sufficient to cause maximal deproteinization of horse serum, as judged by (a) the
157
appearance of the supernatant, and (b) the observation that further additions of sulphosalicylic acid did not produce further precipitation from the supematant. However, it was found necessary to add excess sulphosalicylic acid (final concentration 50 g/l) in order to avoid the formation of a white precipitate when the solution was made alkaline. Colour development The acidic supematant must be made alkaline in order to permit the formation of the paraquat cation radical. Conditions for colour development were tested as follows. Horse serum was treated with an equal volume of 100 g/l sulphosalicylic acid. To the protein-free supernatant were added increasing volumes of alkaline dithionite solution, and the mixture scanned from 460-370 nm in zero-order and second derivative modes. At approximately pH 12 (Universal Indicator Paper, Whatman-B.D.H.) an interfering yellow colour was formed which disappeared on further addition of alkali to pH 14. Thus it was necessary to add 0.25 ml alkaline dithionite to 1.0 ml supematant so that a flat baseline resulted. Similar results were obtained when pooled human plasma was used. Horse serum containing 0.50 mg/l paraquat dichloride was then treated with an equal volume of 100 g/l sulphosalicylic acid. To 1.0 ml of the resultant supernatant was added 0.25 ml alkaline dithionite solution. The mixture was scanned from 460-370 nm in zero-order and in second and fourth derivative modes (Fig. l), using the conditions shown in Table II for the range O.l- 1.0 mg/l. The derivative mode conferred a significant enhancement in sensitivity compared with zero order spectroscopy. The colour proved to be stable in a stoppered cuvette, with no measurable change in derivative amplitude over a two hour period. In the presence of sulphosalicylic acid, the absorbance at 396 nm is 89% of that measured in its absence. (2) The measurement of plasma paraquat The analytical procedure described was applied or plasma.
to the assay of paraquat
in serum
Linearity, precision and recovery In both second and fourth derivative spectra, the derivative amplitude was linearly related to paraquat dichloride concentration over two ranges: O.l- 1.0 mg/l and 1.0-5.0 mg/l, as reported in Table II. In horse serum 0.05 mg/l paraquat dichloride could be reliably detected by either second or fourth derivative spectroscopy. Coefficients of within-batch variation for 0.5 mg/l paraquat dichloride in horse serum were: Second derivative
(n = S), 5.09%
Fourth
(n = 8), 4.70%.
derivative
Comparison with aqueous standards containing sulphosalicylic acid showed that 90% of the paraquat added to horse serum (at 0.50 mg/l) was recovered in the supematant after protein precipitation.
Add to 2 ml horse serum:
Follow method, reference [S]
2tnl loog/l TCA
2 ml 100 g/1 PCA
Knepil technique
Ttichloracetic acid
Perchloric acid
Slight precipitate tends to form on adding NaOH to supematant. liichloroacetate reacts with paraquat cation radical.
0.067
0.123
2.1
3.2
SLigbt precipitate tends to form on adding NaOH to supematant. Perchlorate reacts with paraquat cation radical.
Alkaline supematant sometimes turbid, occasionally causing interference in 2nd derivative spectrum.
0.149
1.4
Yield of protein-free supematant (ml)
Comments
METHODS FOR THE DIRECT ASSAY OF PARAQUAT IN SERUM Absorbance * of alkaline supematant at 396 nm
OF SIX PROTEIN PRECIPITATION
Technique
COMPARBON
TABLE I
43%
Recovery in supematant of added paraquat dichloride **
159
w 8 d
h! m
c? t-4
0,
X
II
0.10-1.00 0.10-1.00 1.0 -5.0 1.0 -5.0
(mg/l)
Concentration range
PERFORMANCE
142 r2.9 236 24.7 37.1 kO.45 22.5-cO.41
Slope 2 SD
OF SECOND
I.722 1.80 2.14i-2.87 -0.622 1.48 -0.32:~ 1.36
SD
DERIVATIVE
Intercept*
AND FOURTH
FOR PARAQUAT
0.5OO-tO.051 0.500=!=0.049 3.00 1-0.13 3.00 -0.20
mg/l mg/l mg/l mg/l
Confidence limits (p=O.95) for stated level of paraquat dichloride
ASSAYS
SERUM
0.9994 0.9994 0.9998 0.9995
Correlation coefficient
IN HORSE
AFTER
5 5 5 5
n
PROTEIN
The derivative peak amplitude was measured graphically with respect to the adjacent satellite peak at higher wavelength, the Pye-Unicam SP S-100 instrumental conditions being optimized for each concentration range and derivative order. Second derivative: scan speed 120 nm/min; spectral band-width I.0 nm; derivative unit time-constant G3 (undamped); absorbance scale 0.5 a.u.f.s. (0.10-1.00 mg/lf or 2.0 a.u.f.s. (I .O-5.0 mg/l). Fourth derivative: scan speed 120 nm/min; spectral band-width I .O nm; derivative unit time constants (undamped) G2 and G3; absorbance scale 0.2 a.u.f.s. (0. IO- I .OO mg/l) or 2.0 a.u.f.s. (1.0-5.0 m&l).
2 4 2 4
Order
QUANTITATIVE PRECIPITATION
TABLE
161
Interferences Potential spectroscopic interferences were investigated by assaying specimens of horse serum, pooled human serum or plasma, and human plasma exhibiting a high bilirubin level (18 1 pmol/l) or severe lipaemia, using the more sensitive instrumental parameters described in Table II. In none of these cases was any significant
r
,
(b) ,
, T
,(c),
405 396
390
430
390
430
390
430
X,nm Fig. 2. Comparison of zero and second derivative spectra after deproteinisation and dithionite reduction of: (a) 0.50 mg/l paraquat dichloride in horse serum; (b) patient’s plasma sample displaying haemolysis to the extent of 2%; (c) 0.50 mg/l paraquat dichloride incorporated in (b). The derivative amplitude D, for the paraquat cation radical peak is unaffected by haemolysis if measured between 390 nm and 396 nm. Instrumental conditions as in Table II (0. lo- I .OOmg/l).
162
contribution to the second or fourth derivative amplitude at the paraquat wavelengths observed. The effect of haemolysis was studied by applying the proposed method to visibly haemolysed plasma (containing no paraquat) from 15 patients. Unprecipitated haeme gave rise to a zero-order peak at 421 nm with a corresponding second derivative peak at 417 nm, whose amplitude correlated approximately with the degree of haemolysis as judged visually. Although the derivative spectrum of each haemolysed sample was zero at 390 nm and 396 nm, the p,resence of a satellite maximum at 405 nm distinct from, but close to, the paraquat satellite maximum at
Second
Derivative
(Protein
Precipitation)
Method
. . .
.
0.
.
r = 0.9946
$/
/.
/
P-
I 1.0
I 2.0 Paraquat
Laboratory Fig. 3. Comparison of paraquat dichloride derivative method after protein precipitation,
Method
I 3.0
I 4.0
mg/ I
: Ion-pair
Extraction
concentration in plasma specimens assayed by the second and by the standard ion-pair extraction method [6].
163
396 nm, could give rise to ambiguity in the measurement of the paraquat derivative amplitude (Fig. 2). Haemolysis was also induced artificially by treating with saponin a known fraction of the cells separated from human plasma and adding back to the plasma. Over the range O-2% haemolysis the amplitude of the derivative peak at 417 nm was approximately linearly related to the degree of haemolysis. This relationship broke down above 2% haemolysis. Plasma samples were prepared in this manner, with haemolysis in the range O-2%, and containing 0.50 mg/l paraquat dichloride. No significant interaction was observed between the haemolysis second derivative spectrum and the measured derivative amplitude of the paraquat, provided that the latter was measured precisely from the peak minimum (390 nm) to the wavelength of its satellite maximum (396 nm) (Fig. 2). Visual comparison showed that nearly all haemolysed plasma samples separated in the routine clinical chemistry laboratory within a few hours of collection fell within the range O-2% haemolysis. Comparison with the ion-pair extraction method 23 samples from poisoned patients within the range 0.05-4.0 mg/l paraquat dichloride were analysed by the protein precipitation technique, using second derivative spectroscopy. All had been analysed on receipt by the standard ion-pair extraction method [6] used in the laboratory. The results shown in Fig. 3 indicate that correlation is good (r = 0.995; slope + SD = 0.975 & 0.021; intercept & SD = 0.034 2 0.031). All results were calculated with reference to a bracketing standard of 1.00 mg/l paraquat dichloride, analysed before and after each batch of specimens. Discussion
Methods for the calorimetric estimation of paraquat oy dithionite reduction require the prior removal from plasma of interfering substances, especially protein. Chemical deproteinization is more rapid than extraction, but the background absorbance of the resultant supematant renders it unsuitable for zero-order spectroscopy. However, second or fourth derivative spectroscopy virtually eliminates this matrix interference and has conferred on the best of the deproteinization techniques a sensitivity and precision comparable to ion-pair extraction. The correct choice of conditions for protein precipitation is essential. Four of the standard techniques (Table I) cannot be used because the reagent is incompatible with the alkaline dithionite detection system, whereas two methods are generally applicable: sulphosalicylic acid precipitation and the technique of Knepil [5]. The Knepil supematant however, was sometimes affected by turbidity, which persisted after addition of alkali; with occasional plasma samples it was so severe as to cause unacceptable interference in the second derivative spectrum. Moreover, in our hands, only 43% of paraquat added to serum could be recovered in the Knepil supematant. The sulphosalicylic acid supematant, on the other hand, was always clear after the addition of alkali. The dilution of the plasma by the reagent was compensated by the 90% recovery of added paraquat in the supematant. The sample requirement could therefore be reduced from 2 ml to 1 ml. Indeed in extreme cases
164
0.5 ml plasma may be treated with 0.5 ml sulphosalicylic acid; sufficient supernatant is obtained for paraquat measurement in a semimicro cuvette after the addition of one-quarter its volume of alkaline dithionite. The absorbance of reduced paraquat at 396 nm is decreased by 11% in the presence of sulphosalicylic acid, but the colour is stable since there are no organic solvent residues to interact with the paraquat cation radical. The potential interference of sample haemolysis can be recognized visually and can be readily overcome by careful measurement of the paraquat second derivative amplitude. An alternative approach would be to measure the amplitude from the peak to the derivative baseline zero (Dz), rather than to the long-wavelength satellite ( DL). This measurement is, however, more susceptible to baseline changes produced, for example, by slight variation in the dithionite concentration. On balance, sulphosalicylic acid deproteinization was considered to be more straightforward and reliable than the Knepil technique for use in a simple emergency procedure to be performed by moderately experienced staff on a 24-h basis. Although the fourth derivative mode Gas shown to be slightly more sensitive and precise than the second, it was felt that these benefits did not justify the increased investment in a further electronic module for routine applications. Moreover, fourth derivative spectra tend to be less easy to interpret than second. The amplitude and shape of the derivative spectra generated by electronic resistance-capacitance modules depend markedly on scan speed and the degree of damping incorporated [ 111. Since these parameters may vary slightly from day to day, the use of a within-run standard is recommended. A single bracketing standard was found to be adequate in view of the good linearity obtained with the equipment described, but this standard should be processed at the beginning and end of each batch of analyses in order to compensate any instrumental drift. Using the method described, it is possible to give a result to the requesting clinician within 20 min of the receipt of a specimen, thereby allowing prompt treatment of the patient. The method easily measures paraquat in the range within which clinical decisions are made [l]. It is anticipated that analogous sample treatment procedures followed by derivative spectroscopic detection could be applied to the analysis of paraquat in tissue samples of human or animal origin. Acknowledgements
The generous loan of equipment for second and fourth derivative spectrophotometry by Pye-Unicam Ltd., Cambridge, UK, for use in this work is gratefully acknowledged. References 1 Proudfoot AT, Stewart MJ, Levitt T, Widdop B. Paraquat poisoning: significance of plasma paraquat concentrations. Lancet 1979; II: 330-332. 2 Levitt T. Radioimmunoassay for paraquat. Lancet 1977; II: 358. 3 Levitt T. Determination of paraquat in clinical practice using radioimmunoassay. Proc Anal Div Chem Sot 1979; 16: 72-76.
165 4 Fatori D, Hunter WM. Radioimmunoassay for serum paraquat. Clin Chim Acta 1980; 100: 81-90. 5 Knepil J. A short, simple method for the determination of paraquat in plasma. Clin Chim Acta 1977; 79: 387-390. 6 Jarvie DR, Stewart MJ. The rapid extraction of paraquat from plasma using an ion-pairing technique. Clin Chim Acta 1979; 94: 241-251. 7 Stewart MJ, Levitt T, Jarvie DR. Emergency estimations of paraquat in plasma. A comparison of the RIA and ion-pair/calorimetric methods. Clin Chim Acta 1979; 94: 253-257. 8 O’Haver TC. Potential clinical applications of derivative and wavelength modulation spectrometry. Clin Chem 1979; 25: 154% 1553. 9 Fell AF. Derivative spectroscopy in the analysis of aromatic amino acids. In: Rattenbury JM, ed, Amino acid analysis, Chapter 6. Chichester and New York: Ellis Horwood-John Wiley, 1981: 86-118. IO Fell AF, Jarvie DF:, Stewart MJ. Analysis for paraquat by second- and fourth-derivative spectroscopy. Clin Chem 1981; 27: 286-292. 11 Fell AF. Analysis of pharmaceutical dosage forms by second derivative ultraviolet-visible spectrophotometry. Proc Anal Div Chem Sot 1978; IS: 260-267.
Clinica Chimica Actu, 117 (1981) 153-165 Elsevier/North-Holland Biomedical Press
CCA
1946
A rapid method for the emergency analysis of paraquat in plasma using second derivative spectroscopy D.R. Jar-vie a, A.F. Fell b,* and M.J. Stewart
’
a Toxicology
Unit, Department of C&al Chemistry, Royal Infirmary, Edinburgh EH3 9Y W, ’ Department of Pharmacy, Heriol- Watt University, Edinburgh EHI 2HJ and ’ Department of Clinical Biochemistry, Royal Infirmary, Glasgow G4 OSF (UK) (Received
April 16th. 1981)
A rapid method for the analysis of paraquat in plasma of poisoned patients is described. The dithionite colour reaction is used to detect paraquat, and matrix interference is eliminated by the use of a chemical deproteinization technique followed by second derivative spectroscopy of the reaction mixture. The method was compared with an established procedure using ion-pair extraction followed by dithionite reduction. Sensitivity and precision were comparable and the correlation in patients’ plasma was good. The proposed direct method is rapid and suitable for emergency use in the management of patients poisoned with .paraquat.
Introduction
The prognosis for the patient severely poisoned with paraquat is poor, unless the poison can be eliminated from the body before’ it causes irreversible damage to sensitive tissues. Techniques such as haemodialysis and haemoperfusion which remove paraquat from the plasma space must be instituted as early as possible, while the plasma concentration is high, if they are to have any likelihood of success [l]. Methods for the measurement of plasma paraquat concentration as a basis for treatment must therefore be both rapid and precise. Rapid radioimmunoassay methods are available [2-41, but this technique is not always suitable for use in the general clinical chemistry laboratory. Of the calorimetric assays based on dithionite reduction of paraquat, that of Knepil [5] is rapid but subject to interference from endogenous materials in plasma. It is therefore unsuitable for accurate determina* To whom correspondence OC09-8981/81
should be addressed.
/OOOC-0000/$02.75
0 1981 Elsevier/North-Holland
Biomedical
Press
154
tion at the lower levels, around 0.1 mg/l, .at which clinical decisions may be required [l]. Such interference is eliminated by ion-pair extraction in the method of Jarvie and Stewart [6,7], which has given good service in recent years for the care of poisoned patients in our respective hospitals. This method has adequate sensitivity, but requires good pipetting technique and takes about 40 min for the complete analysis of a specimen. Derivative spectroscopy (8- 1 l] offers an alternative approach to the reduction of background absorbance. In this mode the normal (zero-order) absorption spectrum is transformed to a function of its second (d2A/dA2) or higher even derivative, by an analog or digital device as the spectrum is scanned. Such modules are available as relatively inexpensive accessories for several well-known commercial spectrophotometers, so that the technique is well within the scope of the smaller laboratory. In derivative mode, sharp features such as the paraquat cation radical peak are enhanced, to give improved resolution of overlapping bands, whereas broader absorption bands such as those arising from non-specific matrix absorption are suppressed. Recent studies have characterized the behaviour of the second and fourth derivative spectra of the paraquat cation radical [lo]. Derivative detection confers four major advantages over the zero order mode. The interference due to diquat, often co-formulated in commercial weedkiller preparations, is almost eliminated in derivative mode and the need for baseline correction is therefore avoided. Slight differences in dithionite concentration between test and reference cuvettes seriously disturb the zero-order absorption spectrum, but have less effect in derivative mode, thus conferring increased precision at lower paraquat concentrations. The interference of endogenous substances is reduced, implying that extraction may be replaced by a simpler purification stage. Because of the enhanced sensitivity in the derivative mode, a concentration step is no longer required. The present work describes the development and evaluation of a rapid method of paraquat estimation in plasma by derivative spectroscopy after protein precipitation and dithionite reduction. The method has a detection-limit of 0.05 mg/l paraquat dichloride, is simple to perform and is suitable for emergency use. Materials and methods Instrumentation A model SP8-100 scanning spectrophotometer, equipped with two electronic derivative modules (Pye-Unicam Ltd., Cambridge, UK), was used with a matched pair of quartz semimicro cuvettes of. 1 cm path-length (Pye-Unicam Ltd). Simple modification to isolate the microswitch actuating an optical filter at 390 nm was necessary to eliminate the electrical interference spike on the derivative spectrum. Derivative spectroscopy The second and fourth derivative spectra of the absorption peak of the paraquat cation radical at 396 nm were generated using the optimized instrumental conditions summarized in the legend to Table II. The derivative amplitudes were measured
155
from peak to long-wavelength satellite (see Fig. 1); this measurement has been shown to be more sensitive and precise in paraquat analysis than measurement from peak to derivative baseline zero [lo]. The effect of random noise was reduced by recording each derivative spectrum three times and using the average derivative amplitude for analytical calculations. In the initial investigations of linearity and precision, both second and fourth derivative spectra were examined. Since the fourth derivative was found to give comparable results and conferred little advantage in terms of precision and sensitivity in the present work, second derivative measurements alone were used for the analysis of patients’ samples. Paraquat standard
Paraquat dichloride (Aldrich Chemicals, Poole, Dorset, UK) was dried to constant weight before use (2 h at 1OO’C) and subsequently stored in a desiccator. Stock solutions (20 and 200 mg/l) were prepared in distilled water, and serum standards made from them by dilution in horse serum (Horse Serum 3, Wellcome Reagents Ltd., Becker&am, Kent, UK).
Fourth
Second
Zero
I
A-
I
I
I
I
t
t
0.05A
I.021
+
1
I
DL
Lt
I
Ii
DL
i
Dz
I I
390
430
390
430
I
Dz
’ Is-
r t
390
430
h,nm Fig. 1. Zero order, second and fourth derivative spectra of 0.50 mg/l paraquat dichloride in serum after deproteinization with sulphosalicylic acid and reduction with alkaline dithionite. Instrumental conditions as in Table II (0.10-1.00 mg/l). The graphical measures available are: D,, peak to long wavelength satellite; D,, peak to derivative baseline zero. The paraquat cation radical peak is observed at 396 nm (zero order), 390 run (second derivative) and 387 nm (fourth derivative) due to the time constants of the electronic differentiators employed, which lead to a wavelength shift in the direction of scanning.
156
Reagents (1) Sulphosalicylic
acid, 100 g/l. A solution of 5-sulphosalicylic acid (Analar, B.D.H. Chemicals Ltd., Poole, Dorset, UK) in distilled water. (2) Alkaline dithionite solution, 20 g/l. 0.2 g sodium dithionite was dissolved in 10.0 ml aqueous 5 mol/l NaOH immediately before use in a batch of analyses. The reagent is stable for approximately 2 h.
Patients’ specimens All samples analysed for paraquat were taken from poisoned patients admitted during the previous 12 months to the Regional Poisoning Treatment Centre, Royal Infirmary of Edinburgh. Paraquat-free samples for the study of spectral interference were collected locally in the routine clinical chemistry laboratory. Blood was collected in tubes containing lithium heparin as anticoagulant. Plasma was separated on receipt in the laboratory, analysed for paraquat by the standard method [6] and then stored at -20°C pending repeat analysis by the protein precipitation method.
Analytical procedure To 1.0 ml serum or plasma in a lo-ml plastic tube add 1.0 ml 100 g/l sulphosalicylic acid solution. Mix thoroughly (10 s) by vortex mixer and centrifuge at 1500 X g for 5 min in a bench centrifuge. Decant the supernatant (total vol. 1.6 ml). To 1.0 ml of the supernatant in a second plastic tube add 0.25 ml alkaline dithionite solution. Mix thoroughly and transfer to a 1.0 cm seminicro quartz cuvette. Record the second derivative spectrum using the conditions described in Table II and scanning from 430 to 370 nm, using as reference a fresh mixture of 1.0 ml distilled water and 0.25 ml alkaline dithionite solution. Process a paraquat dichloride standard (1 .OO mg/l in horse serum) in the same manner as the test samples and analyse before and after each batch of tests. Measure the derivative amplitude in tests and standards from the peak minimum at 390 nm to the long-wavelength satellite maximum at 396 nm (mean of 3 scans). If haemolysis is present, measure to the precise wavelength of the long-wavelength satellite, as recorded in the spectrum of the 1.00 mg/l paraquat standard. The paraquat dichloride concentration in the test samples is calculated by linear proportion. Results
(I) Development of the analytical method Choice of protein precipitant Five standard techniques of protein precipitation were applied to horse serum (Table I). The technique specifically designed for paraquat determination by Knepil [5] was also tested. Of these six techniques, only sulphosalicylic acid proved reliable enough to form the basis of an analytical method. Similar results were obtained using pooled human plasma. A final sulphosalicylic acid concentration of 25 g/l was sufficient to cause maximal deproteinization of horse serum, as judged by (a) the
157
appearance of the supernatant, and (b) the observation that further additions of sulphosalicylic acid did not produce further precipitation from the supematant. However, it was found necessary to add excess sulphosalicylic acid (final concentration 50 g/l) in order to avoid the formation of a white precipitate when the solution was made alkaline. Colour development The acidic supematant must be made alkaline in order to permit the formation of the paraquat cation radical. Conditions for colour development were tested as follows. Horse serum was treated with an equal volume of 100 g/l sulphosalicylic acid. To the protein-free supernatant were added increasing volumes of alkaline dithionite solution, and the mixture scanned from 460-370 nm in zero-order and second derivative modes. At approximately pH 12 (Universal Indicator Paper, Whatman-B.D.H.) an interfering yellow colour was formed which disappeared on further addition of alkali to pH 14. Thus it was necessary to add 0.25 ml alkaline dithionite to 1.0 ml supematant so that a flat baseline resulted. Similar results were obtained when pooled human plasma was used. Horse serum containing 0.50 mg/l paraquat dichloride was then treated with an equal volume of 100 g/l sulphosalicylic acid. To 1.0 ml of the resultant supernatant was added 0.25 ml alkaline dithionite solution. The mixture was scanned from 460-370 nm in zero-order and in second and fourth derivative modes (Fig. l), using the conditions shown in Table II for the range O.l- 1.0 mg/l. The derivative mode conferred a significant enhancement in sensitivity compared with zero order spectroscopy. The colour proved to be stable in a stoppered cuvette, with no measurable change in derivative amplitude over a two hour period. In the presence of sulphosalicylic acid, the absorbance at 396 nm is 89% of that measured in its absence. (2) The measurement of plasma paraquat The analytical procedure described was applied or plasma.
to the assay of paraquat
in serum
Linearity, precision and recovery In both second and fourth derivative spectra, the derivative amplitude was linearly related to paraquat dichloride concentration over two ranges: O.l- 1.0 mg/l and 1.0-5.0 mg/l, as reported in Table II. In horse serum 0.05 mg/l paraquat dichloride could be reliably detected by either second or fourth derivative spectroscopy. Coefficients of within-batch variation for 0.5 mg/l paraquat dichloride in horse serum were: Second derivative
(n = S), 5.09%
Fourth
(n = 8), 4.70%.
derivative
Comparison with aqueous standards containing sulphosalicylic acid showed that 90% of the paraquat added to horse serum (at 0.50 mg/l) was recovered in the supematant after protein precipitation.
Add to 2 ml horse serum:
Follow method, reference [S]
2tnl loog/l TCA
2 ml 100 g/1 PCA
Knepil technique
Ttichloracetic acid
Perchloric acid
Slight precipitate tends to form on adding NaOH to supematant. liichloroacetate reacts with paraquat cation radical.
0.067
0.123
2.1
3.2
SLigbt precipitate tends to form on adding NaOH to supematant. Perchlorate reacts with paraquat cation radical.
Alkaline supematant sometimes turbid, occasionally causing interference in 2nd derivative spectrum.
0.149
1.4
Yield of protein-free supematant (ml)
Comments
METHODS FOR THE DIRECT ASSAY OF PARAQUAT IN SERUM Absorbance * of alkaline supematant at 396 nm
OF SIX PROTEIN PRECIPITATION
Technique
COMPARBON
TABLE I
43%
Recovery in supematant of added paraquat dichloride **
159
w 8 d
h! m
c? t-4
0,
X
II
0.10-1.00 0.10-1.00 1.0 -5.0 1.0 -5.0
(mg/l)
Concentration range
PERFORMANCE
142 r2.9 236 24.7 37.1 kO.45 22.5-cO.41
Slope 2 SD
OF SECOND
I.722 1.80 2.14i-2.87 -0.622 1.48 -0.32:~ 1.36
SD
DERIVATIVE
Intercept*
AND FOURTH
FOR PARAQUAT
0.5OO-tO.051 0.500=!=0.049 3.00 1-0.13 3.00 -0.20
mg/l mg/l mg/l mg/l
Confidence limits (p=O.95) for stated level of paraquat dichloride
ASSAYS
SERUM
0.9994 0.9994 0.9998 0.9995
Correlation coefficient
IN HORSE
AFTER
5 5 5 5
n
PROTEIN
The derivative peak amplitude was measured graphically with respect to the adjacent satellite peak at higher wavelength, the Pye-Unicam SP S-100 instrumental conditions being optimized for each concentration range and derivative order. Second derivative: scan speed 120 nm/min; spectral band-width I.0 nm; derivative unit time-constant G3 (undamped); absorbance scale 0.5 a.u.f.s. (0.10-1.00 mg/lf or 2.0 a.u.f.s. (I .O-5.0 mg/l). Fourth derivative: scan speed 120 nm/min; spectral band-width I .O nm; derivative unit time constants (undamped) G2 and G3; absorbance scale 0.2 a.u.f.s. (0. IO- I .OO mg/l) or 2.0 a.u.f.s. (1.0-5.0 m&l).
2 4 2 4
Order
QUANTITATIVE PRECIPITATION
TABLE
161
Interferences Potential spectroscopic interferences were investigated by assaying specimens of horse serum, pooled human serum or plasma, and human plasma exhibiting a high bilirubin level (18 1 pmol/l) or severe lipaemia, using the more sensitive instrumental parameters described in Table II. In none of these cases was any significant
r
,
(b) ,
, T
,(c),
405 396
390
430
390
430
390
430
X,nm Fig. 2. Comparison of zero and second derivative spectra after deproteinisation and dithionite reduction of: (a) 0.50 mg/l paraquat dichloride in horse serum; (b) patient’s plasma sample displaying haemolysis to the extent of 2%; (c) 0.50 mg/l paraquat dichloride incorporated in (b). The derivative amplitude D, for the paraquat cation radical peak is unaffected by haemolysis if measured between 390 nm and 396 nm. Instrumental conditions as in Table II (0. lo- I .OOmg/l).
162
contribution to the second or fourth derivative amplitude at the paraquat wavelengths observed. The effect of haemolysis was studied by applying the proposed method to visibly haemolysed plasma (containing no paraquat) from 15 patients. Unprecipitated haeme gave rise to a zero-order peak at 421 nm with a corresponding second derivative peak at 417 nm, whose amplitude correlated approximately with the degree of haemolysis as judged visually. Although the derivative spectrum of each haemolysed sample was zero at 390 nm and 396 nm, the p,resence of a satellite maximum at 405 nm distinct from, but close to, the paraquat satellite maximum at
Second
Derivative
(Protein
Precipitation)
Method
. . .
.
0.
.
r = 0.9946
$/
/.
/
P-
I 1.0
I 2.0 Paraquat
Laboratory Fig. 3. Comparison of paraquat dichloride derivative method after protein precipitation,
Method
I 3.0
I 4.0
mg/ I
: Ion-pair
Extraction
concentration in plasma specimens assayed by the second and by the standard ion-pair extraction method [6].
163
396 nm, could give rise to ambiguity in the measurement of the paraquat derivative amplitude (Fig. 2). Haemolysis was also induced artificially by treating with saponin a known fraction of the cells separated from human plasma and adding back to the plasma. Over the range O-2% haemolysis the amplitude of the derivative peak at 417 nm was approximately linearly related to the degree of haemolysis. This relationship broke down above 2% haemolysis. Plasma samples were prepared in this manner, with haemolysis in the range O-2%, and containing 0.50 mg/l paraquat dichloride. No significant interaction was observed between the haemolysis second derivative spectrum and the measured derivative amplitude of the paraquat, provided that the latter was measured precisely from the peak minimum (390 nm) to the wavelength of its satellite maximum (396 nm) (Fig. 2). Visual comparison showed that nearly all haemolysed plasma samples separated in the routine clinical chemistry laboratory within a few hours of collection fell within the range O-2% haemolysis. Comparison with the ion-pair extraction method 23 samples from poisoned patients within the range 0.05-4.0 mg/l paraquat dichloride were analysed by the protein precipitation technique, using second derivative spectroscopy. All had been analysed on receipt by the standard ion-pair extraction method [6] used in the laboratory. The results shown in Fig. 3 indicate that correlation is good (r = 0.995; slope + SD = 0.975 & 0.021; intercept & SD = 0.034 2 0.031). All results were calculated with reference to a bracketing standard of 1.00 mg/l paraquat dichloride, analysed before and after each batch of specimens. Discussion
Methods for the calorimetric estimation of paraquat oy dithionite reduction require the prior removal from plasma of interfering substances, especially protein. Chemical deproteinization is more rapid than extraction, but the background absorbance of the resultant supematant renders it unsuitable for zero-order spectroscopy. However, second or fourth derivative spectroscopy virtually eliminates this matrix interference and has conferred on the best of the deproteinization techniques a sensitivity and precision comparable to ion-pair extraction. The correct choice of conditions for protein precipitation is essential. Four of the standard techniques (Table I) cannot be used because the reagent is incompatible with the alkaline dithionite detection system, whereas two methods are generally applicable: sulphosalicylic acid precipitation and the technique of Knepil [5]. The Knepil supematant however, was sometimes affected by turbidity, which persisted after addition of alkali; with occasional plasma samples it was so severe as to cause unacceptable interference in the second derivative spectrum. Moreover, in our hands, only 43% of paraquat added to serum could be recovered in the Knepil supematant. The sulphosalicylic acid supematant, on the other hand, was always clear after the addition of alkali. The dilution of the plasma by the reagent was compensated by the 90% recovery of added paraquat in the supematant. The sample requirement could therefore be reduced from 2 ml to 1 ml. Indeed in extreme cases
164
0.5 ml plasma may be treated with 0.5 ml sulphosalicylic acid; sufficient supernatant is obtained for paraquat measurement in a semimicro cuvette after the addition of one-quarter its volume of alkaline dithionite. The absorbance of reduced paraquat at 396 nm is decreased by 11% in the presence of sulphosalicylic acid, but the colour is stable since there are no organic solvent residues to interact with the paraquat cation radical. The potential interference of sample haemolysis can be recognized visually and can be readily overcome by careful measurement of the paraquat second derivative amplitude. An alternative approach would be to measure the amplitude from the peak to the derivative baseline zero (Dz), rather than to the long-wavelength satellite ( DL). This measurement is, however, more susceptible to baseline changes produced, for example, by slight variation in the dithionite concentration. On balance, sulphosalicylic acid deproteinization was considered to be more straightforward and reliable than the Knepil technique for use in a simple emergency procedure to be performed by moderately experienced staff on a 24-h basis. Although the fourth derivative mode Gas shown to be slightly more sensitive and precise than the second, it was felt that these benefits did not justify the increased investment in a further electronic module for routine applications. Moreover, fourth derivative spectra tend to be less easy to interpret than second. The amplitude and shape of the derivative spectra generated by electronic resistance-capacitance modules depend markedly on scan speed and the degree of damping incorporated [ 111. Since these parameters may vary slightly from day to day, the use of a within-run standard is recommended. A single bracketing standard was found to be adequate in view of the good linearity obtained with the equipment described, but this standard should be processed at the beginning and end of each batch of analyses in order to compensate any instrumental drift. Using the method described, it is possible to give a result to the requesting clinician within 20 min of the receipt of a specimen, thereby allowing prompt treatment of the patient. The method easily measures paraquat in the range within which clinical decisions are made [l]. It is anticipated that analogous sample treatment procedures followed by derivative spectroscopic detection could be applied to the analysis of paraquat in tissue samples of human or animal origin. Acknowledgements
The generous loan of equipment for second and fourth derivative spectrophotometry by Pye-Unicam Ltd., Cambridge, UK, for use in this work is gratefully acknowledged. References 1 Proudfoot AT, Stewart MJ, Levitt T, Widdop B. Paraquat poisoning: significance of plasma paraquat concentrations. Lancet 1979; II: 330-332. 2 Levitt T. Radioimmunoassay for paraquat. Lancet 1977; II: 358. 3 Levitt T. Determination of paraquat in clinical practice using radioimmunoassay. Proc Anal Div Chem Sot 1979; 16: 72-76.
165 4 Fatori D, Hunter WM. Radioimmunoassay for serum paraquat. Clin Chim Acta 1980; 100: 81-90. 5 Knepil J. A short, simple method for the determination of paraquat in plasma. Clin Chim Acta 1977; 79: 387-390. 6 Jarvie DR, Stewart MJ. The rapid extraction of paraquat from plasma using an ion-pairing technique. Clin Chim Acta 1979; 94: 241-251. 7 Stewart MJ, Levitt T, Jarvie DR. Emergency estimations of paraquat in plasma. A comparison of the RIA and ion-pair/calorimetric methods. Clin Chim Acta 1979; 94: 253-257. 8 O’Haver TC. Potential clinical applications of derivative and wavelength modulation spectrometry. Clin Chem 1979; 25: 154% 1553. 9 Fell AF. Derivative spectroscopy in the analysis of aromatic amino acids. In: Rattenbury JM, ed, Amino acid analysis, Chapter 6. Chichester and New York: Ellis Horwood-John Wiley, 1981: 86-118. IO Fell AF, Jarvie DF:, Stewart MJ. Analysis for paraquat by second- and fourth-derivative spectroscopy. Clin Chem 1981; 27: 286-292. 11 Fell AF. Analysis of pharmaceutical dosage forms by second derivative ultraviolet-visible spectrophotometry. Proc Anal Div Chem Sot 1978; IS: 260-267.