amount taken, and a and b are the intercept and slope, respectively. In all cases the confidence interval for the slope includes unity, indicating that the analyses are accurate and consistent over the range covered. Also the confidence interval for the intercept includes zero, from which i t is inferred that in no case is there a blank dependent on the amount of constituent. The estimated standard deviation for bismuth difference would be expected to be somewhat larger than
reported here, because it should contain the errors Of the Other determinations. The fact that it is not larger suggests the presence of compensating errors. However, because the precision was satisfactory, no effort was made to isolate these errors. LITERATURE CITED
(1) Duval, Clement, “Inorganic Thermoravimetric Analysis,” pp. 302, 384, New York, 1953 (2) Fritz, J. S., ANAL. CHEM. 26, 1978 (1954).
blsevier,
(3) Grbnkvist, K. E., Farm. Roy. 52, 305 (1953). (4) Hillebrand, W. F., Lundell, G. E. F., Bright, H, A,, Hoffman, J. I., L‘Applled Inorganic Anal ais,” pp. 280, 334, Wiley, New Yorc, 1953. (5)Theory ofA*Statistics, M., “In!foduction Chaps. 11, to the 13, McGraw-Hill, New York, 1950.
(6) Youden,, W.,,J., “Statistical Methods for Chemists, Chap. 5, Wiley, Kew York, 1951. for review October 19, 1959. RECEIVED Acce ted January 22, 1960. Pittsburgh Conzrence on Analytical Chemistry and A plied Spectroscopy, Pittsburgh, Pa., $arch 1959.
Automated System for Continuous Determination of Penicillin in Fermentation Media Using Hydroxyla mine Reagent A.
0.NIEDERMAYER, F. M. RUSSO-ALESI, C. A. LENDZIAN, and JACQUES M. KELLY’
Squibb lnstitufe for Medical Research, New Brunswick, N. .I.
b An automated colorimetric method for the determination of penicillin based on the reaction of penicillin with hydroxylamine and ferric ion is presented. It is valid in the range of 0 to 10,000 units per ml. and compares satisfactorily with the microbiological diffusion assay. The method has been used extensively in the control of pilot and plant fermentations. The standard error is 2.5%.
A
x AUTOMATED METHOD for the chemical determination of penicillin in fermentation media using the Technicon AutoAnalyzer has been described (4). The chemistry of the method is based on the inactivation of penicillin to penicilloic acid, either enzymatically or chemically (1). The preformed penicilloic acid is separated by means of the continuous dialysis unit of the AutoAnalyzer. Potencies are estimated by colorimetrically measuring the differences in iodine uptake, between active and degraded penicillin. I n initial trials, the method was reasonably satisfactory in most cases. However, its limitations became evident as broader concentration ranges and widely varying fermentation media were studied. Valid analyses of many fermentation media encountered were confined to rather narrow ranges of 1 Present address, Charles Pfizer & Co., Brooklyn, N. Y.
664
ANALYTICAL CHEMISTRY
concentration and necessitated the preparation of aqueous penicillin standards a t levels approximating those of the samples. The best workable concentration range was of the order of magnitude of 400 to 1600 units per ml. Outside this range, pronounced curvature was , observed and, hence, the samples required dilution and reanalysis. Furthermore, with some fermentation media, the responses of serially diluted samples with water were not linear. The reason for this phenomenon is still not clearly understood. However, it can be demonstrated that the dialysis of serially diluted aqueous penicillin standards through the membrane is a curvilinear function. Details of this work will be reported elsewhere. Attempts to use the iodine method without dialysis were unsatisfactory because of gross interferences from broth constituents. Hydroxylamine was first suggested as a reagent for penicillin by Boxer and Everett (8). Penicillin reacts with this reagent and ferric ion to give a colored complex. On the other hand, its alkaline or penicillinase degradation product does not give a colored complex. Therefore, by running a sample and blank determination, the penicillin potency can be estimated from the difference in the color response. The method has been widely used in the fermentation industries. The details of this method have been modified for its automated operation on the AutoAnalyzer.
METHOD OF ANALYSIS
A standard type Technicon AutoAnalyzer is used without the continuous dialyzer. One additional time-delay coil is used in the system to permit adequate inactivation of penicillin by penicillinase. The coil is a glass helix of sufficient length to give a 5-minute contact time. Reagents. PENICILLIN STANDARDS. Dissolve a predetermined weight of potassium benzylpenicillin in sufficient distilled water to give the equivalent of 10,000 units per ml. Dilute this stock standard with distilled water t o give standard solutions of 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, and 10,000 units per ml. Use these levels of concentration t o construct the standard curve. PENICILLINASE. Dissolve 100,000 units of Schenley penicillinase A in 150 ml. of distilled water a t room temperature. Store the reagent in an ice water bath. It is stable under these conditionu for a t least 8 hours. HYDROXYLAMINE REAGENT(STOCK SOLUTION).Dissolve 350 grams of “*OH. HC1 (J. T. Baker) in water and dilute to 1 liter. The reagent is stable for a t least 2 weeks a t room temperature. ALKALIBUFFER (STOCKSOLUTION). Dissolve 173 grams of sodium hydroxide and 20.6 grams of anhydrous sodium acetate in water and dilute to 1 liter. WORKING HYDROXYLAMINE REAGENT. Titrate a portion of the alkali buffer solution against the hydroxylamine hydrochloride solution t o p H 7.0, using a pH meter. To 1 volume of neutralized NHzOH. HC1 solution add 8 volumes of water and 2 volumes of 95y0 ethyl alcohol. This reagent is
stable for a t least 8 hours a t room temperature. FERRICAMMONIUM SULFATEREAGENT (15%). Dissolve 15 grams of FeNH4(S04)2.12Hz0in 70 ml. of 1.ON sulfuric acid. Preparation of Samples and Blanks. Centrifuge 15 t o 25 ml. of penicillin broth a t sufficient speed t o give a clear supernatant fraction. A 10-ml. aliquot of the supernate is adequate t o fill the sample cups of the automatic sampling device for a “blank” and “sample” determination. Flow Diagram. The flow diagram for the method is shown in Figure 1. The numbers adjacent t o the tubes of the manifold refer t o the volumes of fluid and air pumped per minute. For each estimate of potency a blank and sample determination are needed. The sample is initially picked up from the sample plate and segmented by air bubbles through introduction of a continuous stream of air. A stream of distilled mater is admitted to dilute the simple to an accpptable working level. This mixture is pumped through a standard mixing coil (horizontal glass helix) to ensure homogeneity and then through the 5-minute time-delay coil a t room temperature. At the end of this period, it is joined by a continuous stream of hydroxylamine reagent and passes through a second standard mixing coil. After mixing, it meets a continuous stream of ferric ammonium sulfate reagent in a T connection. To prevent deposition of a precipitate that forms a t this point. it is necessary to mount this connection so that the cross member of the T is in the vertical position, The main stream enters through the stem of the T and the ferric ammonium sulfate reagent through the top of the cross member. This mixture is then passed through a 3-minute time-delay coil, which also acts as a mixing coil. a t room temperature to develop the color complex. The colored stream enters the colorimeter, where the transmittance is measured at 480 mp in a 1.0-cm. flow cuvette and recorded. The flow system for the blank determination is the same, except that the penicillinase reagent is substituted for distilled water. The sampling plate is run a t the rate of 40 sample cups per hour. In this method, to allow for adequate cleansing of the system, it is necessary to separate samples (and blanks) with cups of distilled water. This results in a net rate of 10 estimations per hour. It is preferable to run all samples for a given day before the blanks. Penicillinase contaminate8 the tubing and the entire system must be cleaned with 2N hydrochloric acid for 10 pumping minutes after its use, if additional samples are to be run. RESULTS AND DISCUSSION
Using aqueous penicillin standards it can be demonstrated that linearity is achieved over the range of 0 to 10,000 units of penicillin per ml. A typical per cent transmittance plot covering
Y 1.0 II. C.ll
DISCAID
Figure 1. Flow diagram for determination of penicillin with hydroxylamine reagent in AutoAnalyzer
IO 20 Figure 2. Recorded transrnittancies of aqueous penicillin standards
30
W
40
V
z
5
4
E 50 I
0
v,
z 4
E 60 x
-
8 m
s 0 0 0 N
70
\
this range of standards is shown in Figure 2. The standard curve was found to be reproducible from day to day within an over-all error of 2%. In the first evaluation of the method with fermentation media, eight samples of broth, each from a different fermentation tank, were studied, The samples were taken independently in a random order on different days. They were also analyzed independently in a random order at two different times using three levels of concentration of unknowns and two of compamble standards. The concentration levels were adjusted in accordance with the six-point design (2), which specifies that dilutions be of equal logarithmic interval. This facilitates the calculation of parallelism between standards and unknowns, curvature and opposed curvature in re-
sponse and validity. All data were processed on an IBM-650 computer. Actual potencies are given in Table I. Checks were made for slope, parallelism, curvature, and opposed curvature. These calculations showed a very satisfactory agreement with the hypothesis of validity as outlined by Bliss (2). The component of total experimental error introduced by the operation of the assay method a t a given time was represented by a standard error of 2.5%. The actual potencies (Table I) show unusually good agreement, This demonstrates that the performance of the instrument system and the chemical reactions involved in the analysis give a satisfactory degree of precision. It was also necessary to assess its ability to measure actual broth potency. VOL. 32, NO. 6, M A Y 1960
665
This was accomplished by a second experimental design consisting of a crossover experiment with a microbiological diffusion assay (5). Eighteen samples of fermentation broth of various penicillin concentrations were used. Aliquots of the same sample of broth and standards were supplied to two different assay laboratories from a central source. Each laboratory used the sixpoint assay design referred to above. The microbiological assays were run in duplicate. As before, all data were processed on the computer and a number of statistical tests were applied to the data. Table I1 lists the calculated potencies obtained by the two methods. The correlation coefficient (y = 0.9868 or y 2 = 0.9737) shows that the chemical potency accounts for 97.37% of the variability in penicillin activity among the broth samples and that the two methods are highly correlated. The f test for regression shows that the reduction in variation associated with
the regression is highly significant. Therefore, it is concluded that the automated chemical method measures true microbiological potency of penicillin in fermentation media. Experience with the method in actual pilot and plant operations has
Table II. Comparison of AutoAnalyzerHydroxylamine and Microbiological Diffusion Methods for Determination of Penicillin in Fermentation Broths
Broth Sample 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
Table I. Reproducibility of AutoAnalyzer-Hydroxylamine Method for Penicillin
Penicillin Potency, Unitslhfl. Unknown Day 1 Day 2 1 2 3 4 5 6
7
8
3370
3340
2580 4970 3980 3750
2540 4860 3420 3960 4410 ~ ~ 4720 3320
4300 ~~
~
4640 3210
Penicillin Potency, Unite/Ml. AutoMicrobiol. Analyzer diff. Six-Point Design 3440 2060 3330 2140 3840 4140 3340 1090 3050 1480 3950 4450 4490 2630 4410 4530 4110 4730
3340 2270 3520 2270 4360 4650 3860 1090
3260 1630 4300 4980 4580 2900 4610 4470 4180 5190
Single Determinations
_
.
1 2 3 4 5 6 7
2040 3030 4230 250 1530 3020 4860
2100 3000 4000 110 1440 3050 4720 ~~
~
shown that it is also of value as a method for single determination analysis. In this case, the potency is obtained by subtracting the blank from the unknown after first converting both to penicillin potency from a standard curve. Comparable chemical and microbiological results using single determination analyses are also given in Table 11. I n addition to the extension of the range of analyses, the method eliminates the undesirable maintenance features connected with the use of the dialyzer unit. ACKNOWLEDGMENT
The authors are indebted to D. R. Embody, Electronic Data Processing Center, for statistical analyses and data processing and to Joseph Levin, Microbiological Assay Laboratories, for cooperation in the crossover studies. The technical assistance of Constance Sherman and Edith McGrath is also gratefully ackcowledged, LIT~~ATURE CITED
(1) Alicino, J. F., ISD. E m . CHEJI., A K A L ED. . 18, 619 (1946). (2) Bliss, C. I., “Statistics of Bioassay,”
reprinted ,yith additions from “Vitamin Methods, Vol. 11, pp. 445-628, Acndemic Press, New York, 1952. (3) Boxer, G. E., Everett, P. M., ANAL. CHEW21,670 (1949). (4) Ferrari, A,, Russo-Alesi, F. hI., Kelly, J. hf., Ibid., 31, 1710 (1959). (5) Grove, D. C., Randall, W. A , , “Assay of Antibiotics, Laboratory Manual,” Medical Encyclopedia, New York 22, N.Y., 1955.
RECEIVED for review August 21, 1959. Accepted Janiiary 19, 1960.
Use of a Modified Ehrlich‘s Reagent for Measurement of lndolic Compounds MARJORIE KNOWLTON, F. CURTIS DOHAN,’ and HERBERT SPRlNCE Veferans Adminisfration Hospital, Coatesville, Pa.
b A modification of the Ehrlich benzaldehyde reaction for measurement of indole and indole derivatives has been developed. By the use of a nearly saturated solution of p-dimethylaminobenzaldehyde in 12N hydrochloric acid, the sensitivity and stability of the Ehrlich reaction have been greatly improved. Micromolar concentrations of indolic compounds may b e measured with accuracy and precision.
T
isolation and quantitative identification of compounds containing HE
666
ANALYTICAL CHEMISTRY
1
the indole nucleus have recently been intensively studied because of their possible involvement in disease processes. Some indoles can be measured satisfactorily (6,6); others, with difficulty. Brief reviews of the theory and use of the Ehrlich benzaldehyde reaction with indoles have recently been published (8, 9 ) . There is diversity of opinion as to the choice of acid and the concentration of the reagent. Earlier studies from this laboratory (4) set forth the factors influencing this reaction and emphasized the
importance of the ratio of benzaldehyde concentration to acid concentration. Preliminary studies with a commonly used reagent of 2% p-dimethylaminobenzaldehyde (PDAB) in 12N hydrochloric acid revealed the transitory nature of the reaction, the violet color changing to green or blue, often with the formation of a colloidal precipitate. The effects of the independent variation of concentration of hydrochloric acid 1
Present address, William Pepper Lab-
oratory of Clinical Medicine, University of Pennsylvania, Philadelphia, Pa.
