Table I. Sulfate D a t a on I.A.P.S.O. Seawater Sulfate, ppt'
i
J
Figure 1. Polarogram of a standard seawater solution prepared as described In the text. Shape of the curve lndlcates no Interferences in seawater samples
After recording, the electrodes were well rinsed with distilled water and wiped dry. A starting potential of 0.2 volts was used and the solutions were degassed with dry nitrogen for two minutes prior to recording. A full scale sensitivity of 10 r A was used. A polarogram of a seawater solution with excess lead(I1) nitrate is shown in Figure 1. All seawater samples were I.A.P.S.O. Standard Seawater with an adjusted chlorinity of 19.375 ppt. All standard seawater ampoules were from the same hatch. The constancy of ratios of major ions in seawater indicates that the sulfate concentration should be 2.712
2 -702 2.732 2.725 2.693 2.700 2.720 2.720 2.750 2.735 2.696 Mean 2.717 i 0.010 Standard deviation 0.70% a The average of two runs on the same samples.
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3/13/75 4/11/75 4/11/75 4/17/75 4/17/75 4/18/75 4/18/75 4/28/75 4/28/7 5 4/28/75
duce the seawater volume required. Preliminary data also indicate that sulfate concentrations in the vicinity of 200 ppm are easily measurable, allowing the technique to be applicable to brackish waters. In such cases, a determination of the chloride content indicated that a corresponding increase in the volume of samples should be utilized because the standards had been prepared to a specific ionic strength. The combination of rapidity, precision, and small sample volume makes this technique extremely desirable.
PPt.
ACKNOWLEDGMENT R E S U L T S A N D DISCUSSION
As an indication of' the accuracy of the technique, the correlation coefficients for the calibration curves indicate a precision of 0.2% or less. The data for the standard seawater samples (Table I) have a range from 2.693 f 0.010 to 2.750 & 0.010 ppt and a mean value of 2.717 f 0.010 ppt. The standard deviation of the measured samples is 0.70%. The precision o f the technique indicates its suitability for sulfate analysis. The technique appears not to be subject to any interferences normally present in seawater samples. When fully operational, this technique can process 25-30 samples in a five-hour period. New polarographic instrumentation now appearing on the market should further increase the rapidity of the technique. Simple modification of the sample preparation can substantially re-
We thank A. Galuppo and 0. Stepowyj for providing careful checks on operator error by independently performing the technique. LITERATURE CITED (1) H. J. Kelly and L. B. Rodgers, Anal. Chem.,27, 759 (1955). (2) M. R. McSwain, R. J. Watrous, and J. E. Dougiass, Anal. Chern., 46, 1329 (1974). (3) "Technicon Auto Analyzer Methodology". Technicon Corporation, Tarrytown, N.Y., Method No. 226-72W, 1969. (4) J. Ross and M . Fraut, Anal. Chem., 41, 967 (1969). (5) R. Jasinski and I. Trachtenberg, Anal. Chem.,44, 2373 (1972). (6) G.S. Elen, Anal. Chern., 26, 909 (1954). (7) 0. I. Miine, Anal. Chem., 24, 1247 (1952). (8) C. S.Martens and R. A . Berner, Sclence, 185, 1167 (1974).
RECEIVEDfor review June 23, 1975. Accepted July 10, 1975.
Trace Level Determination of 1,4-Benzodiazepines in Blood by Differential Pulse Polarography M. A. Brooks and M. R. Hackman
Department of Biochemistry and Drug Metabollsm, Hoffmann-La Roche Inc., Nutley, N.J. 071 10 The new generation of highly efficacious drugs, which are often administered in doses as low as 1 to 2 mg, requires highly sensitive techniques capable of the measurement of compounds in biological fluids in the nanogram g) range. The measurement of drugs a t these low levels is commonly performed using gas chromatography with an electron-capture detector or by spectrofluorometry and, more recently, by radioimmunoassay and mass fragmentography. Sensitive polarographic analysis has recently become possible through advances in instrumentation which en-
ables the analyst to perform fast scan and various forms of pulse polarography. The use of differential pulse polarography (DPP) to measure drugs in biological fluids has been reported (1). D P P has recently been applied to the determination of trimethoprim and its metabolites in blood and urine (2, 3 ) , phenobarbital, diphenylhydantoin ( 4 ) , several nitroimidazoles (5) and glibornuride in blood (6),and to the measurement of the urinary excretion of several 1,4benzodiazepines (7-9). Chlordiazepoxide and its metabolites in human plasma have been measured with an absolute limit of detection of 50-100 ng/2 ml plasma (IO).
ANALYTICAL CHEMISTRY, VOL. 47, NO. 12, OCTOBER 1975
2060
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The determination of two 1,4-benzodiazepines a t an absolute limit of detection of 10-20 ng/ml using a new polarographic microcell and miniaturized electrodes will be presented to demonstrate the utility of DPP for trace analysis.
EXPERIMENTAL
Suitable aliquots of the working solutions are added to blood as internal standards. Extraction Procedure. To a 15-ml conical centrifuge tube, add 1 ml of blood and 2 ml of pH 12.8 saturated trisodium phosphate buffer. Extract with 2 X 5 ml of benzene:methylene chloride (90: 10). Transfer the extracts to another 15-ml conical centrifuge tube and evaporate to dryness under a stream of nitrogen. Dissolve the residue in 0.5 ml of the appropriate supporting electrolyte, viz., 1M phosphate buffer (pH 4.0) for N-desalkylflurazepam and 1M phosphate buffer (pH 7.0) for bromazepam. Deaerate the sample by bubbling nitrogen through the sample for approximately 1 minute. Transfer the deaerated sample to the polarographic cell and analyze using the polarographic parameters previously described. Calculations. The currents resulting from reduction of the 4 3 azomethine bond common to both N-desalkylflurazepam and bromazepam are measured a t -0.725 and -0.610 V vs. Ag/AgCl, respectively. The overall recoveries and concentrations are determined as described previously ( 2 ) .
Instrumentation. All experiments were performed using a Princeton Applied Research Corporation (P.A.R.) Model 174 Polarographic Analyzer equipped with a P.A.R. Model 172A Drop Timer and electrode assembly and a Houston Omnigraphic X-Y recorder, (Model 2230-3-3). Electrode Assembly. A three-electrode semimicro cell with a working volume of 0.5 ml containing a dropping mercury electrode [DME: 5-in. length of common spirit white back thermometer tubing of outside diameter 0.130 f 0.005 inch (3.30 f 0.13 mm) with a 6 5 - g bore ( m = 2.67 mg/sec) from Corning Glass Works] as the indicator electrode, a Ag/AgCl electrode (fiber junction from Microelectrodes, Inc., Londonderry, N.H., No. MI-401) as the reference electrode, and a platinum wire (B&S 22 gauge) wrapped around the Ag/AgCl reference as the auxiliary electrode was used (see Figure 1). RESULTS AND DISCUSSION I n s t r u m e n t a l Parameters. Polarography was performed in the Cell Design. The microcell (Figure 1)was designed with differential pulse mode with a -100-mV pulse being applied using a 2.0-second drop. The flow rate was 2.67 mg/sec (m2'3t1'6 = a flared top for easy transfer by pouring of a sample from a 2.1603). Scans were performed from -0.600 to -0.850 V vs. Ag/ 15-ml conical centrifuge tube, and with a stopcock a t the AgCl for N-desalkylflurazepam and from -0.500 to -0.750 V vs. bottom to facilitate saving the sample after analysis. It was Ag/AgCl for bromazepam a t a scan rate of 1 mV/sec with a full also designed to prevent the immediate shorting of the scan range of 1.5 V. The current sensitivity required ranged from electrodes by the accumulated waste mercury. Using a 0.5 t o 5 pA full scale deflection. DME having a drop mass m = 2.67 mghec, approximately Blood Assays. Reagents. All reagents were of analytical reagent grade (ACS) purity and were used without further purification ex5 minutes can be used for each scan before the mercury cept as indicated otherwise. The buffer used in the initial extracbuild up shorts out the electrodes. This allows for a scan of tion was saturated trisodium phosphate (pH 12.8). The reagent 300 mV a t a rate of 1 mVlsec. Therefore, it is essential that was made by adding 200 g of NaSP04 to 500 ml of distilled water the electrodes be set just into the upper portion of the 0.5and shaking until saturated. ml solution away from the mercury buildup a t the bottom The supporting electrolytes for D P P analysis were 1M phosof the cell. phate buffers of p H 4.0 and p H 7.0 containing 0.005% methoxy polyethylene glycol 550 (Matheson, Coleman & Bell, Fine ChemiApplications. Comparison of Relative Sensitivities of cal Grade) prepared as follows: phosphate buffer (pH 4.0) is pret h e 2.0- and 0.5-ml Capacity Cells. In order to evaluate the pared by mixing 420 ml of 1 M KH2P04 and 80 ml of 1M absolute limit of detection of the new microcell, a compariand adjusting to pH 4.0 with 1M KH2P04 or 1 M &Po4 solution, son was made to the 2-ml cell used in our previous studies whereas the p H 7.0 buffer is prepared by mixing 195 ml of 1M (7, IO). Duplicate standards of N-desalkylflurazepam were KHzP04 and 305 ml of 1M KzHP04. T o each solution add 2.5 ml prepared in the concentrations of 10, 20, 50, 100, 200, 500 of 1%aqueous solution of methoxy polyethylene glycol 550. Standard Solutions. Dissolve 10.0 mg of N-l-desalkylflurazepand 1000 ng/2.0 and 0.5 ml of the 1M phosphate buffer (pH am [7-chloro-5-(2-fluorophenyl)-1,3-dihydro-2~-1,4-benzodi- 4.0), for polarographic analysis in the 2.0- and 0.5-mi cells, azepin-2-one, C15HloNzOClF (mol wt = 288.5; m p = 205-206 OC)] respectively. The data obtained were normalized for slight and bromazepam [7-bromo-5-(2-pyridyl)-1,3-dihydro-2H-l,4-bendifferences in bore size of the DME using their respective zodiazepin-2-one, C14H10N30Br (mol wt = 316.16, m p = 237-238.5 m 2 / 3values and are shown in Figure 2. The data indicated "C)] in 100 ml of methanol to give separate stock solutions, each that polarographic reduction of the compound in both cells containing 100 gg/ml. Dilute 0.10 ml of each stock solution to 100 ml with methanol to give working solutions containing 100 ng/ml. yielded a linear calibration throughout the working concen2060
ANALYTICAL CHEMISTRY, VOL. 47, NO. 12, OCTOBER 1975
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zepam Table I. Blood Levels of N-1-Desalkylflurazepam Following Chronic Administration of a Single Daily 30-mg O r a l Dose of Dalmane Concentration, n g l m l
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Concentration
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EC-GLC
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19 24 24 20 30 24 33
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Figure 5. Internal and external calibration curves of bromazepam
Table 11. Blood Levels of Bromazepam Following Chronic Administration of a Single Daily 3-mg Oral Dose Concentration, n g / m l
tration range. The decrease in volume of the supporting electrolyte from 2.0 to 0.5 ml resulted in a corresponding fourfold increase in the absolute level of detection. With this increase in detection level, the lo-, 20-, and 50-ng samples were measurable in the microcell, while the same amounts of materials were nonmeasurable using the 2-ml cell. The absolute limit of detection is based on the measurement of a peak of height = 1.0 cm a t a current range of 0.5 WAfull scale deflection. Analytical measurements a t a current range of 0.2 WAfull scale deflection were not feasible because of interferences from the reduction of impurities in the supporting electrolyte. DPP Analysis of 1,4-Benzodiazepines in Blood. The analysis of two 1,4-benzodiazepines in blood was investigated to test the utility of the micropolarographic cell for low level determinations of drugs in biological fluids. The D P P behavior of this series of compounds has been described ( 1 1 ) . The primary blood metabolite of flurazepam-hydrochloride (Dalmane) in man is N-desalkylflurazepam (12). Following chronic administration of 30 mg of Dalmane, levels of flurazepam and hydroxyethylflurazepam are typically below 5 ng/ml of blood while levels of N-desalkylflurazepam are usually between 25-50 ng/ml of blood (8). The levels of parent drug and hydroxyethyl metabolite are below
DaY
Polamgraphic
1 4 9 10 12 15
26 48 69 71 123 119
EC-GLC
27 53 92
77 125 105
the sensitivity limit of the polarographic assay, whereas those of N-desalkylflurazepam are readily determined. Blood samples taken from day 2 to 7 were analyzed from a subject who received 30 mg of Dalmane daily for 14 days according to the polarographic procedure described above. The assay has a recovery of 70.6% f 5.8 (std dev) in the range of 10-1000 ng of N-desalkylflurazepam/ml of blood. Internal and external calibration for N-desalkylflurazepam curves are shown in Figure 3. The samples were also analyzed using the same extraction procedure by electron capture-gas liquid chromatography (EC-GLC) ( 8 ) , however, employing a 3% OV-17 (4 ft, 4-mm i.d.) column, at an oven temperature of 235 “C. Under these conditions, N-desalkylflurazepam had a retention time of 5.2 minutes. The data in Table I show that analyses using both techniques are in good agreement. However, the EC-GLC assay is to be preferred because of its higher degree of specificity and its capability of determining levels down to 2-3 ng/ml of blood.
ANALYTICAL CHEMISTRY, VOL. 47, NO. 12, OCTOBER 1975
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Following the administration of bromazepam, no metabolites are found in the blood (7); therefore, D P P (see Figure 4) can be used without employing thin-layer chromatography to ensure specificity. Blood samples taken a t 6 hours on days 1, 4, 9, 10, 12, and 15 from a subject who received an oral dose of 3 mg of bromazepam daily were analyzed by the polarographic assay described above. The assay has a recovery of 62.1% f 7.8 (std dev) in the range of 10-1000 ng bromazepam/ml of blood. Internal and external calibration curves for bromazepam are shown in Figure 5 . The same samples were also analyzed employing the EC-GLC previously reported ( 7 ) . The data in Table I1 show good agreement between the two assays. Although the chromatographic assay is more specific than the polarographic assay, both assays have essentially the same absolute limit of detection.
CONCLUSIONS Using the conventional type of cell, the absolute limit of detection of differential pulse polarographic analysis for two 1,4-benzodiazepines was increased to 10 ng of compound by miniaturization of the cell and its operational electrodes. Further increases in detection limit will only be possible through radical changes in cell design, choices of electrodes, and more sophisticated instrumentation, incorporating signal processing to increase the signal-noise ratio.
ACKNOWLEDGMENT The authors extend thanks to J. Arthur F. de Silva for his critical review of this manuscript, and to Robert McGlynn for the drawings of the figures presented.
LITERATURE CITED (1) M. A . Brooks, J. A. F. de Silva, and M. R . Hackman, Amer. Lab., 5(9), 23-38 119731 (2) M. A. Brooks, J. A. F. de Sllva, and L. M. D'Arconte, Anal. Chem., 45, 263-266 (1973). (3) M. ~ A .Brooks, J, A. F. de Silva, and L. D'Arconte, J. Pharm. Scl., 82, 1395-1397 (1973). (41 M. A . Brooks, J. A. F. de Silva, and M. R. Hackman, Anal. Chlm. Acra. 84, 165-176 (1973). (5)J. A. F. de Silva, N. Munno, and N. Strojny, . . J. Pharm. Sci., 59, 201-210 (1970). (6)J. A . F. de Silva, and M. R. Hackman, Anal. Chem., 44, 1145-1151 (1972). (7) J. A . F. d e Silva, I. Bekersky, M. A. Brooks, R . E. Welnfeld, W. Qlover, and C.V. Pugllsl, J. Pharm. Scl,, 63, 1440-1445 (1974). (8) J. A. F. de Silva, C. V. Puglisl, M. A. Brooks, and M. R . Hackman, J. Chromatogr.,99, 461-483 (1974). 19) J. A. F. de Sllva. I. Bekerskv. and M. A. Brooks, J. Pharm. Scl., 83, 1943-1945 (1974). M. R. Hackman, M. A. Brooks, J. A. F. de Silva, and T . S Ma, Anal. Chem., 48, 1075-1062 (1974). M. A. Brooks, J. J. Bel Bruno, J. A. F. de Silva, and M . R. Hackman, Anal. Chlm. Acta, 74, 367-385 (1975). S. A. Kaplan, J. A . F. de Silva, M. L. Jack, K. Alexander, N. Strojny, R . E. Welnfeld, C.V. Pugllsi, and L. Welssman, J. Pharm. Scb, 62. 19321935 (1973).
__ _ _
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RECEIVEDfor review March 18, 1975. Accepted June 16, 1975. Presented a t the 26th Annual Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy, Cleveland, Ohio, March 3-7, 1975.
Determination of Submicrogram Amounts of Atmospheric Particulate Sulfur Janja D. Husar, Rudolf B. Husar, and Pamela K. Stublts Air Pollution Research Laboratory, Department of Mechanical Engineering, Washington University, St. Louis, Mo. 63 130
The rate and formation mechanism of particulate sulfur in urban atmospheres is one of the intriguing problems of current environmental research. From the point of view of analytical chemistry, the study of sulfate formation in power plant and urban plumes is made difficult because of the short sampling times and consequently small amounts of particulate matter available for analysis. Thus, a strong need exists for an accurate means for determination of particulate sulfur in submicrogram quantities. One of the promising techniques for this purpose is thermal decomposition of particulate sulfur compounds to gaseous products and subsequent detection by flame photometric detector (FPD). The first to use this approach were Scaringelli and Rehme ( 1 ) who slowly heated and vaporized the sample in a furnace. Roberts and Friedlander (2) placed filter sections between two stainless steel strips and rapidly vaporized the sulfur compounds by capacitor discharge. We have applied the technique for the sulfur analysis of filter samples collected in plumes by aircraft as part of the St. Louis Regional Air Pollution Study (RAPS). In the course of the aircraft sample analyses, a need arose for detailed documentation of the technique, to improve its reproducibility, and to lower the detection limit beyond those reported by earlier investigators. 2062
In this paper, we report the adapted procedures for routine sample analyses, calibration data for a variety of sulfur compounds believed to exist in the atmosphere, sensitivity, precision, sample recovery, and comparison results with X-ray fluorescence (XRF).
EXPERIMENTAL Reagents. Reagent grade chemicals were used throughout this study without further purification. Apparatus. The sulfur determining system is shown schematically in Figure 1. It consists of a flash vaporization vessel, flame photometric detector, electronic integrator, and a strip chart recorder. The sample vaporization was performed by capacitor discharge (0.38 F, 12 V) across a tungsten boat, resulting in resistance heating to 1100 OC. Commercially available tungsten boats (E. Fullam Inc., Schenectady, N.Y. 12301, Catalog number 1213, for electron microscope evaporator supplies) were utilized. Proper electrical contact between the supporting metal posts and the boat was ensured by two sets of bolts. The vaporization vessel that houses the tungsten boat was designed to withstand large rates of temperature change. For that purpose, the metal posts that support the tungsten boat were sealed in the bottom part of the vaporization vessel by glass to metal seals (Latronics Co., Latrobe, Pa. 15650), both having the same coefficient of thermal expansion. Vaporized gaseous decomposition products of sulfur compounds were carried to the flame photometric detector by a stream of clean, charcoal filtered air at a flow rate of 2 cm3/sec. The volume
ANALYTICAL CHEMISTRY, VOL. 47, NO. 12, OCTOBER 1975
