Automated Colorimetric Determination of Penicillin in Fermentation Samples Using a Molybdoarsenic Acid-Mercuric Chloride Reagent Kaj Andre Holm Novo Terapeutisk Laboratorium AIS, Ndr. Fasanvej 215, 2200 Copenhagen N , Denmark An automated, simplified, ultramicro method for the continuous determination of penicillins V and G, based on Pan’s fundamental work, i s described. Penicillin is hydrolyzed enzymatically by penicillinase into the corresponding penicilloic acid, which, in the presence of mercuric chloride, has a reducing effect on molybdoarsenic acid. The intensity of the molybdenum blue color which develops is measured. The simplification consists of an omission of the very time-consuming shaking procedure which Pan’s method involved, and in the use of penicillinase conversion of penicillin into penicilloic acid. The reagent concentrations were modified in automating the procedure. The automation results in an analysis with a high degree of specificity, increased accuracy and precision (M = 8764, S% = 0.9), as well as a greater speed of analysis when compared with the manual method described in the text. The sensitivity is about 1 pg of penicillin V per ml.
ONEOF THE FOLLOWING three methods is generally used for the determination of penicillin: the iodine method, described by Goodall and Davies (I), which has a low specificity and is performed with an unstable iodine reagent, the hydroxylamine method, described by Niedermayer et a[. (2), in which the greatest disadvantages are the low sensitivity (about 20 pg of penicillin per ml) and the instability of the resulting color, and the molybdoarsenic acid-mercuric chloride method described by Pan (3). The advantages of this method are: high sensitivity, by which it is possible to measure 1 pg of penicillin V per ml. (This corresponds to the sensitivity of the iodine starch method.) ; high specificity; and stable reagents. Green and Monk (4) developed an automated methodology based on Pan’s original method. Katz and Winnett 1962 (5) and Katz (6) used Pan’s method for analyzing procaine penicillin contained in premixes and mixed feeds by extracting with methanol. Stone (7) employed a new extracting material, chloroform dimethyl formamide, for the determination of procaine penicillin in mixed feeds. Common to all these modifications is the fact that the extraction procedure is retained while only the organic extracting solvents are changed. The method to be described is an automated conversion of an unpublished manual modification of Pan’s work developed in our laboratories. Pan found that alkaline hydrolysis of penicillin gave penicilloic acid, which at room temperature reduced molybdoarsenic acid in the presence of mercuric chloride. The time-consuming solvent extractions, which Pan’s method employs, and the evaporation of the organic (1) R. R. Goodall and R. Davies, Analyst, 86, 326 (1961). (2) A. 0. Niedermayer, F. M. Russo-Alesi, C. A. Lendzian, and J. M. Kelly, ANAL.CHEM., 32,664 (1960). (3) S. C. Pan, ibid., 26, 1438 (1954). (4) N. C. Green and P. R. Monk, Chem. Znd. (London), 78, 1210 (1959). (5) S. E. Katz and A. G. Winnett, J. Agr. Food Chem., 10, 284 (1962). (6) S. E. Katz, J. Ass. Ofic.Agr. Chem., 46,429 (1963). (7) L. R. Stone, ibid., 48, 702 (1965).
phase which contains the penicillin is replaced by dilution of the filtered fermentation sample using phosphate buffer pH 7.5 as the diluent. In addition, the conversion of penicillin into penicilloic acid in this modification is made specifically by penicillinase [Ford (S)]. According to Katz and Winnett (5), the principle of the molybdoarsenic acid-mercuric chloride method is as follows : Penicillin is hydrolyzed either basically or enzymatically into the corresponding penicilloic acid which, in the presence of mercuric chloride, is converted into the corresponding penaldic acid and penicillamine. Penicillamine is oxidized during the formation of a disulfide which results in a reduction of molybdoarsenic acid to molybdenum blue. The intensity of the blue color is measured at 800 nm against a corresponding blank as an expression of the concentration of penicillin present. For the sake of clarity, our simplified manual modification of Pan’s work will be described briefly before proceeding to the automated conversion of the method. The Manual Modification. THEREAGENTS.A: Penicillin V and G standard stock solutions containing 5000 and 7500 units per ml are prepared by dissolving the penicillins in McIlvaines citrate-phosphate buffer pH 6.2. The working solutions are prepared by diluting the stock solutions 1 :50 with phosphate buffer pH 7.5. B : The phosphate buffer pH 7.5 is made by dissolving 5 grams of sodium dihydrogen phosphate (NaH2P04.2H20)and 40 grams of disodium phosphate (Na2HP04.2H20) in one liter of demineralized water. C: The mercuric chloride stock solution contained 0.7 gram of HgC12per liter. D: The molybdoarsenic acid reagent is prepared by dissolving 50 grams of ammonium molybdate [(NH4)6Mo,024.4Hz0] in 700 ml of demineralized water and adding to this 42 ml of concentrated sulfuric acid with cooling and 6 grams of sodium arsenate (NazHAsOd.7H20) dissolved in 60 ml of demineralized water. Finally, demineralized water is added to bring the final volume up to one liter. The solution is kept at 37 “C for at least 24 hours before use. The molybdoarsenic acid-mercuric chloride reagent is prepared for immediate use by mixing the molybdoarsenic acid stock solution and the mercuric chloride stock solution in 1. E: The penicillinase reagent (Penase proportion 1 Leo) contained 2 million Leo units in 100 ml of phosphate buffer pH 7.5. Procedure. The sample is diluted 1:50 with phosphate buffer pH 7.5. To 1.2 ml of this solution, 6.5 ml of phosphate buffer pH 7.5 and 50 pl of penicillinase reagent are added. This mixture is allowed to stand for exactly five minutes before the addition of 1.2 ml of molybdoarsenic acid-mercuric chloride reagent. After a color developing period of exactly 15 minutes, a photometer reading is made. Standard dilutions of 100 and 150 units of penicillin per ml are analyzed exactly like the diluted sample. If both penicillins V and G
+
(8) J. H. Ford, Znd. Eng. Chem., 19,1004(1947). ANALYTICAL CHEMISTRY, VOL. 44, NO. 4, APRIL 1972
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TUBE SIZE (ml/rnin)
in inches
Sa"
PHOSPWE BUFFER
Can' (39)
PHOSPHATE BUFFER
DILUTED SAMPLE PENICILLINASE
DILUTED SAMPLE
-
PHOSPHATE BUFFER 'MOLYBOOARSENIC ACID-R
\
2a035(aL2)MERCURICCHLORIDE-R PRO PORT IONING PUMP I1
Figure 1. Flow diagram of the automated modification smc: single mixing coil, dmc: double mixing coil, w: waste
are analyzed, standards of both penicillins are included. A sample blank and a penicillin standard blank are treated in the same way as the sample, except that the addition of the penicillinase reagent is omitted. Measurements are made in a photometer at 800 nm by reading sample against sample blank and standard against standard blank. The concentration values of the samples are calculated in proportion to the corresponding standard values (penicillin V and G, respectively). The reproducibility of a diluted standard with an average of 100 units per ml ( N = 40) is expressed by the relative standard coefficient 2.3 within the same day. For day-to-day variations, the relative standard coefficient is 4.3 %.
x
EXPERIMENTAL
The Automated Modification. The automation of the manual method described above results in an increased speed of analyses. It is possible to analyze 50 samples per hour in the AutoAnalyzer system compared to about 15 samples per hour by the manual method, the accuracy and precision of analysis being of a higher degree than that obtained with the manual method. Apparatus. The AutoAnalyzer system consists of the following components: A Technicon sampler I1 and proportionating pump I1 fitted with clear Tygon tubing; a double time delay coil in a water bath kept at room temperature; two Beckman 1211 photometers with 800-nm IL-filters and equipped with a Helma 178 flow cell having plane parallel sides, a 2-mm aperture, and a 10-mm light path (Helma Miillheim, Baden, Germany), and a ServolRiter 2,2-pen-recorder (Texas Instruments, USA). Reagents for the AutoAnalyzer Method. A : Standard solutions of potassium penicillin V and sodium penicillin G are made to contain 2500, 5000, 7500, 10000, and 15000 units per ml dissolved in citrate-phosphate buffer pH 6.2. 796
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ANALYTICAL CHEMISTRY, VOL. 44, NO. 4, APRIL 1972
By freezing them to - 15 "C in disposable plastic test tubes, they can be kept for at least one month. B: McIlvaines citrate-phosphate buffer pH 6.2. C : Phosphate buffer pH 7.5 is made by dissolving 5 grams of sodium dihydrogen phosphate (NaH2P04.2H20)and 40 grams of disodium phosphate (Na2HP04.2H20) in one liter of demineralized water. D: The mercuric chloride reagent containing 0.175 gram of HgC12 per liter can be kept for an unlimited time. E: The molybdoarsenic acid reagent is prepared by dissolving 30 grams of ammonium molybdate [(NH4)6M01024. 4Hz0] in 400 ml of demineralized water, and adding to this 25 ml of concentrated sulfuric acid with cooling and 3.6 grams of sodium arsenate (NazHAs04.7Hz0) dissolved in 30 ml of demineralized water. Finally, demineralized water is added to bring the final volume up to one liter. The reagent must be kept at 37 'C for at least 24 hours before use and can be stored indefinitely in a brown bottle. F: The penicillinase reagent (Penase Leo) contains 100,000 Leo units of enzyme in one liter of phosphate buffer pH 7.5. This can be stored up to 5 days by being kept in a brown bottle at room temperature. Procedure. The flow diagram in Figure 1 shows the manner in which the AutoAnalyzer manifold is constructed and includes the number of milliliters per minute passing through the single pump tubes. Forty microliters of sample is diluted 150 times, in two steps, with phosphate buffer pH 7.5. One volume of diluted sample is delivered to the sample side and a corresponding volume to the blank side. Penicillinase is then added to the sample side and phosphate buffer pH 7.5 to a corresponding pump tube located on the blank side and an incubation period in the double mixing coil ensues. The penicilloic acid which is formed during this period subsequently reduces the molybdoarsenic acid-mercuric chloride reagent with initial color development taking place in a single mixing coil and in a single time delay coil placed in a water bath at room temperature. The intensity of the molybdenum blue color is measured at 800 nm.
t
1. OOO
I'mo 0 . 0
0.800
0.000
am 0,W
Ipi
2
BE
o'Mo
3 o.rm
O'=
0.400
0.XY)
0.m
0.200
0.W
zsw
5ow
TUX)
m m
15m
zsw
PENICILLIN-V UNITS/ml
Figure 2. Influence of variations in the molybdoarsenic acid concentration upon the absorption values of different penicillin V standards 100% molybdoarsenic acid reagent 80 % molybdoarsenic acid reagent A 60,70 molybdoarsenic acid reagent 0 50 molybdoarsenic acid reagent X 25 molybdoarsenic acid reanent 0
z z
5 m
7500
PENICILLIN-V
mom
15m
UNlTS/ml
Figure 3. Assay with variation in the mercuric chloride concentration in relation to the absorption values of different penicillin V standards 0 100 mercuric chloride reagent 50%mercuric chloride reagent A 25 mercuric chloride reagent 0 10 mercuric chloride reagent X 5 mercuric chloride reagent
z z z z
The construction of the AutoAnalyzer manifold shows that the sample and sample blank are analyzed simultaneously. The system for analyzer blanks makes use, initially, of the same equipment, including pump tubes, as that for analyzing samples but it is first after the dilution step that the two systems become independent of each other. The results are recorded about 9 minutes after the sample has been picked up by the sampler. The samples are calculated in relation to their respective standards (penicillin V or G ) . The difference in absorption between sample and corresponding sample blank is correlated by means of standard values to the number of units per milliliter. RESULTS AND DISCUSSION
When the manual method was being adapted to the AutoAnalyzer system, it posed great difficulties because of a strong molybdoarsenic acid precipitation in the system. This required a modification of the reagent concentrations. Selection of Molybdoarsenic Acid Concentration. During the experiments with penicillin V standards (0-15000 units per ml) the mercuric chloride concentration (0.175 gram of HgClz per liter) and the penicillinase concentration (100,000 units per liter) were kept constant, while the molybdoarsenic acid concentration was varied. This last mentioned reagent at 100% contains 50 grams of ammonium molybdate and 6 grams of sodium arsenate per liter. As shown in Figure 2, there was an absorption maximum at 60 and 70%. Molybdoarsenic acid concentrations over and below these values gave lower absorption values. For routine use, 60% molybdoarsenic acid was chosen.
Figure 4. Strip chart showing absorption values of penicillin V standards including standard curve, precision study (standard 5000 repeated ten times) and steady state registration Values in units of penicillin V per milliliter Selection of Mercuric Chloride Concentration. During the experiments to find a suitable mercuric chloride concentration, the concentrations of molybdoarsenic acid (60 %) and penicillinase (100,000 units per liter) were kept constant. The reagent at 100% concentration contains 0.175 gram of mercuric chloride per liter. When concentrations of 5 and 10% mercuric chloride were used, there obviously was a ANALYTICAL CHEMISTRY, VOL. 44, NO. 4, APRIL 1972
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Table I. Dilution Experiment on Four Penicillin V Fermentation Samples Dilution
V,
v1
a Undiluted 11600 5800 1+1 3950 1+2 1 +4 2400 a: units per milliliter found;
Va
b a b 11600 12850 12850 6500 13000 11600 11850 4340 13020 12000 2640 13200 b: units per milliliter corrected for dilution.
a 10150
v4
b
10150 loo00 10020 loo00
5000 3340 2000
a 16200 8250 5400 3200
b 16200 16500 16200 16ooo
Table 11. Recovery Experiment Units per ml penicillin V added
V2
v1
d
C
Vn
d
C
0 1900 1000 2381 4350 4281 3500 3381 4545 6450 6445 5650 5545 6522 8350 8422 7600 7522 8333 10050 10233 9250 9333 c: units per milliliter experimental; d: units per milliliter theoretical,
mo
800
900
WAVELENGTH IN nm
Figure 5. Absorption curve illustrating the absorption maximum for a penicillin V standard of 7500 units per ml
O'0. * loo
t
I
)(1
1M
60
1M
TIME IN MINUTES
Figure 6. Color stability of a penicillin V standard of 7500 units per ml expressed by the absorption values in relation to the incubation time deficiency of mercuric chloride. Higher absorption values were found with 25 and 50% concentrations of the reagent than with a 100% concentration, but the standard curves were not quite straight. When a 100% concentration of mercuric chloride was used, the absorption values of the penicillin V standard curve (0-15000 units per milliliter) were according to Beer's law. Therefore, this concentration was selected for 798
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C
1300 3850 6OOo
7850 9550
v4
d
C
d
3681 5845 7822 9633
1400 3650 6050 7950 9700
3781 5945 7922 9733
routine use. Figure 3 shows the way in which the absorption values of penicillin V varied according to the mercuric chloride concentration used. The absorption values of penicillin G standards were found to be equal to those of corresponding penicillin V standards. Selection of Penicillinase Concentration, The penicillinase reagent was made twice as concentrated as was actually necessary to convert 15000 units of penicillin per milliliter to penicilloic acid at the given temperature and time of incubation. The double concentration was selected to ensure that penicillinase was constantly in excess, even though an eventual decrease in the concentration might occur as a result of being kept at room temperature. These concentrations of the reagents were such that the reagent base line showed no change from its initial value after a three-hour run in the AutoAnalyzer. The absorption values of penicillin V standard are given in Figure 4. Absorption Maximum. As shown in Figure 5 there is an absorption maximum at 790-825 nm. For routine use, 800 nm was chosen. Stability of the Molybdenum Blue Color. Figure 6 shows that by the manual method, the maximum color intensity appears 12-18 minutes after the addition of the molybdoarsenic acid-mercuric chloride reagent. Later, the color intensity decreases linearly so that a reduction of about 19 % is found after 3 hours. In the AutoAnalyzer system, about 7 minutes elapses from the addition of the color reagent until the sample enters the photometer flow cell. At that time, the absorption value on the ascending part of the curve is very close to the maximum value. The maximum value is not completely reached, but this is of minor importance since the process is timed exactly in the AutoAnalyzer system. Dilution Experiments. Four different penicillin V fermentation samples were diluted in varying proportions with demineralized water and analyzed. The results which are shown in Table I can be considered as being satisfactory as the fluctuations fall within the accuracy of the analysis. Accuracy. Recovery experiments were made on four different penicilllin V fermentation samples. The results are shown in Table 11. Calculation of the correlation coefficient between the experimental and theoretical results showed that r = 0.994.
Precision. On the same day, a single penicillin V fermentation sample of a series was analyzed 71 times. The following results were obtained: Average value of 8764 units per milliliter with a relative standard deviation of 0.9%. Dayto-day ( N = 50) analyses of penicillin V fermentation samples gave an average value of 7710 units per milliliter with a relative standard deviation of 2.4%. The AutoAnalyzer Method us. the Manual Method. The two methods agreed with a correlation coefficient of 0.97 ( N = 50). Interference. Only a few substances are able to reduce a molybdoarsenic acid-mercuric chloride reagent at room temperature. Among these are strongly reducing salts such as ferro and stanno salts, as well as ascorbic acid and hydroquinone. These substances were not found in any measurable concentrations in the fermentation substrates which were used. However, degradation products of penicillin, such as penicilloic acid, penilloic acid, penillic acid, and penicillamine may be found. Penicilloic acid, the active agent in the method, reacts with molybdoarsenic acid-
mercuric chloride t o the greatest extent. Penilloic acid interfered slightly (less than 10 %), whereas penillic acid and penicillamine exerted strong interference, e.g., 10 mg of penicillamine per milliliter gave a reading corresponding t o 10000 units of penicillin G per milliliter. However, this interference did not affect the final calculated penicillin concentration, since the blank corrected for interference of this nature. Conclusion. The AutoAnalyzer modification of Pan’s procedure results in a method with a high order of specificity, accuracy, precision, and speed of analysis, as well as a high sensitivity. In addition, only very small amounts of sample are required for each analysis. ACKNOWLEDGMENT
The author is very grateful to P. W. Hansen for his valuable help during the preparation of this manuscript. RECEIVED for review September 12, 1971. Accepted December 7,1971.
Indirect Spectrophotometric Determination of Oxalate Using Uranium and 4-(2-Pyridylazo) resorcinol Robert E. Neasl and John C. Guyon2 Department of Chemistry, University of Missouri, Columbia, Mo. 65201 A method that exhibits operational simplicity is presented for the indirect spectrophotometric determination of the oxalate ion. The diminishment in absorbance effected by oxalate on the red uranium(1V)-4(2-pyridylazo)resorcinol (PAR) complex allows oxalate determination at the ppm level in the presence of many foreign ions. The diminishment in absorbance at 515 nm is linear with oxalate in the range 0 to 3 ppm in the final solution.
VERYFEW PROCEDURES are available for the determination of the oxalate ion in parts-per-million concentrations. The methods that do currently exist are essentially limited to spectrophotometric measurements. The major existing systems and their undesirable aspects may be summarized as follows. The direct iron(II1)-oxalate method ( I ) is insensitive and subject to a variety of interferences. An indirect iron(IIIF3hydroxy-1-p-sulfanatophenyl-3-phenyltriazene system (2) cannot tolerate fluoride, phosphate, citrate, and tartrate. The indirect copper(I1)-benzidine approach (3) is very insensitive, includes tartrate, formate, and citrate as serious interferences and furthermore, benzidine is carcinogenic (4). An indirect Present address, Department of Chemistry, Western Illinois University, Macomb, Ill. 61455 Present address, Department of Chemistry, Memphis State University, Memphis, Tenn. ( 1 ) T. Nozaki, F. Hori, and H. Kurhiara, Nippon Kugaku Zusshi, 82,713 (1961). (2) G. Mehra and N. C. Sogani, J. Indian Chem. SOC.,39, 145 (1967). (3) Z . D. Draganic, Anal. Chim. Acta, 28, 394 (1963). (4) T. G. Whiston and G. W.Cherry, Anulysr, 87,819(1962).
chloranilic acid scheme ( 5 ) presents temperature, time, and pH as critical variables and suffers from serious interferences by citrate and tartrate. The systems utilizing scandium(II1)monochromium Bordeaux C and zirconium(IV)-2-carboxybenzene-3,4-dihydroxybenzene(6) are subject to numerous interferences. The use of 4-(2-pyridylazo)resorcinol as an analytical reagent for uranium was first suggested by Pollard, Hanson, and Geary in 1958 (7). In 1963, Florence and Farrar published a method for the spectrophotometric determination of uranium with PAR at pH 8.0 using triethanolamine as buffer (8). Sommer, Ivanov, and Novotna published both a modification of this method using triethanolamine and a method using formate buffer or 20-30% vjv DMF at pH 3.6 (9). At the lower pH, the interference of rare earths and a number of anions is decreased but the interference due to oxalate isgreatly enhanced. This was the basis for preliminary laboratory studies to determine whether this interference could be utilized in a method for oxalate. No previous use of the uranium(1V) interaction with 4-(2pyridy1azo)resorcinol (also referred to as PAR) to determine oxalate was found. This paper reports the experimental results of work performed in the development of a new simple, ( 5 ) J. de Oliveira Meditsch, Eng. Quim., 15(8), 9 (1963). (6) N. V. Zaglyadimova and Z. M. Gur’eva, Tr. Khim. Khim. Teknol., 1,119 (1967). (7) F. H. Pollard, P. Hanson, and W. J . Geary, Anal. Chim. Acta. 20,26 (1959). ( 8 ) T. M. Florence and Y.Farrar, ANAL. CHEM., 35, 1613 (1963). (9) L. Sommer, V. M. Ivanov, and H. Novotna, Tulunta, 14, 329 ( 1967). ANALYTICAL CHEMISTRY, VOL. 44, NO. 4, APRIL 1972
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