Boll. Chim. Farm.

, 145 (1):4, 2005, CD-ROM

COMPUTERIZED CALCULATION OF BIOLOGICAL POTENCY OF ANTIBIOTICS: INTERACTIVE COMPUTER PROGRAM '' POTENCY v.2'' (TEST:''5+3'' DOSES): CALCULATION OF BIOLOGICAL POTENCY OF NEOMYCIN AND BACITRACIN ANTIBIOTICS IN ENBECIN OINTMENT

Milan B. Arambai and Radmila Mandi

Pharmaceutical factory '' GALENIKA a.d''., Quality Control Center, Dep. Biological Control, YU-11080 Beograd-Zemun, Batajniki put b.b., Serbia and Montenegro

Contact person: Milan B. Arambai Pharmaceutical factory '' GALENIKA a.d''. Quality Control Center Department of Biological Control YU-11080 Beograd-Zemun Batajniki put b.b. Serbia and Montenegro tel. ++ 381 11 30 71 213 fax. ++ 381 11 30 71 076 E-mail: milan.arambasic@hotmail.com

REZIME IZRAUNAVANJE BIOLOKE AKTIVNOSTI ANTIBIOTIKA POMOU RAUNARA: PRIKAZ INTERAKTIVNOG RAUNARSKOG PROGRAMA ''AKTIVNOST v. 2 '' (TEST ''5+3'' DOZE): IZRAUNAVANJE BIOLOKE AKTIVNOSTI ANTIBIOTIKA NEOMICINA I BACITRACINA U ENBECIN MASTI U radu je prikazan interaktivni raunarski program ''AKTIVNOST v. 2'', napisan u programskom jeziku BASIC, na osnovu algoritma za kvantifikovanje rezultata biolokih eksperimenata primenom testa ''5+3'' doze. Izvravanje programa je prikazano na test-primeru izraunavanja bioloke aktivnosti antibiotika neomicina i bacitracina u Enbecin masti (''GALENIKA a.d.'', Beograd). SOMMARIO CALCOLARE DELL' ATTIVITA' BIOLOGICA DEGLI ANTIBIOTOCI MEDIATE IL COMPUTER: LA PRESENTAZIONE DEL PROGRAMMA INTERATTIVO DI COMPUTER ''ATTIVITA v.2 '' (TEST ''5+3'' DOSI): CALCOLARE DELL' ATTIVITA' BIOLOGICA DEGLI ANTIBIOTICI NEOMICINA E BACITRACINA NELL' UNGUENTO ENBECIN Nell' elaborato stato presentato il programma interattivo di computer ''ATTIVITA v. 2'' scrito in lingua di programma BASIC in base all' algoritmo per la quantificazione dei resultati degli esami biologici con l'applicazione del test ''5+3'' dosi. L'esecuzione del programma stata presentata all'esempio del calcolare delle attivit biologiche di antibiotici neomicina e bacitracina nell'inguento Enbecin (''GALENIKA a.d.'', Beograd). SUMMARY This paper presents an interactive computer program ''POTENCY v.2'' written in BASIC program language, based on the algorhythm for quantification of biological test results using ''5+3'' dose test. The program execution is presented by an example of the calculation of biological potency of antibiotics neomycin and bacitracin in the medicinal product ENBECIN ointment (''GALENIKA a.d.'', Beograd). Keywords: BASIC program; Biological assay; Microbiological assay; Diffusion method; Zone of inhibition; Assay ''5+3'' doses; Biological potency; Ointment; Antibiotic; Neomycin; Bacitracin

INTRODUCTION Quality control of starting materials and finished medicinal products can be: chemical, physico-chemical and biological. Biological control includes the assessment of biological potency of antibiotics using the methods of diffusion on agar and turbidimetry. Experimental results comprise a great number of numerical data that should be processed using suitable statistical methods both to evaluate the biological potency of a specified antibiotic and to assess experiment reliability (error limit of the experiment). Both the biological potency of a tested antibiotic and the limit of error of experiment can be calculated manually (based on the existing algorhythms), but a computerized procedure makes the calculation significantly faster and simpler. This paper presents an interactive computer program ''POTENCY v.2'' written in BASIC program language, based on the algorhythm for quantification of biological test results using ''5+3'' dose test. The program execution is presented by an example of the calculation of biological potency of antibiotics neomycin and bacitracin in the medicinal product ENBECIN ointment (''GALENIKA a.d.'', Beograd) using microbiological assay (diffusion method). THEORETICAL DESCRIPTION OF THE PROGRAM POTENCY v.2 The program is designed in such a way to request basic identification to be entered first: name of the tested product (substance), its declared potency and batch number; name of the standard solution, its potency and batch number (code) and, finally test date. The microbiological assay (diffusion method) is based on the comparison of sensitive microorganism inhibition zones obtained with identical standard and test concentrations (1,2). Since the test is carried out with a great number of Petri plates, due to potentially different agar thickness, inhibition zones obtained with identical antibiotic concentrations (standard or sample) may differ in size. To avoid this problem, inhibition zones are subjected to correction by entering first the inhibition zone values obtained with the following concentrations: S1S3, S2S3, S4S3, S5S3, T2S3, T3S3 and T4S3. Based on this information, the program is able to calculate the mean value for inhibition zone diameters obtained with the reference standard concentration S3. Then, based on this, it corrects the values for inhibition zone diameters obtained with standard concentrations S1, S2, S4 and S5 and test concentrations T2, T3 and T4 (3). The program is interactive since, to execute it, it is necessary to enter the following information: 1) values for the standard concentrations S1, S2, S3, S4 and S5; 2) assumed (declared) potency of the tested product. As neither Yugoslav Pharmacopoeia(1) nor European Pharmacopoeia(2) define which statistical method sholud be used for experimental data evaluation (calculate the potency using appropriate statistical methods), program ''POTENCY v.2'' was designed applying algorhitm for quantification of biological assays data, wich fulfill

the requirements of the US Pharmacopoeia: use a log transformation, straight-line method with least squares fitting procedure, and a test for linearity (4). After the values of standard concentrations S1- S5 have been entered, based on the pairs of experimental values for X(i) (the values for standard concentrations S1 (X1), S2 (X2), S3 (X3), S4 (X4) and S5 (X5) and the value for Y(i) (the corrected values for inhibition zone diameters made considering the standard concentrations S1 (Y1), S2 (Y2), S4 (Y4), S5 (Y5) and the reference one S3 (Y3) using the logarithm function with a free member Y = a + b * log X (Eq.1) as the mathematical model which perform linearization of experimental data using log transformation of indipendend variable X only (semi-log transformation), the program determines the values for the following parameters:a (Y-axis intercept) and b (line slope) of linear regression, the relevant value for square error (E), coefficient of correlation (R) and the value for Student test (T) for the calculated coefficient of correlation. Further, based on the parametric values (a and b) obtained by experimental data processing obtained for the set YOBS values (corrected zone inhibition values based on test concentrations T2, T3 and T4) the program calculates XCALC (the relevant test concentration values T2, T3 and T4) and compares them (calculates percentual deviation) to the set values for test concentrations T2, T3 and T4. Then the mean deviation value (%) for calculated test concentrations T2, T3 and T4 is calculated starting from the set T2, T3 and T4 values and, based on the declared potency of the test preparation, calculates the biological potency of the tested antibiotic in appropriate units.

RESULTS Results of the application of the ''POTENCY v. 2 '' program (execution of assayexamples measure of program correctness). Neomycin (5) The standard solution was prepared in the following concentrations (in I.U./ml): 6.4 (S1), 8 (S2), 10 (S3), 12.5 (S4) and 15 (S5), while the test sample (assumed potency: 3300 I.U./g) was prepared in concentrations (in I.U./ml): 8 (T2), 10 (T3) and 12.5 (T4). Each concentration was tested on 3 agar plates each with 6 holes. Three holes in each Petri plate were filled with the reference standard concentration S3 and the remaining three holes were filled either with one of the standard or sample concentrations. The test was done twice from a single homogenous sample. Staphylococcus aureus ATCC 6538-P was used as the test organism. Monitored parameter: size of diameter of inhibition zones (in mm).

FIRST TEST: GROUP S1S3 20.5 20.4 20.8 20.6 20.4 20.6 20.4 20.2 20.3 NUMBER OF SAMPLES S1S3 = 9 SUM OF SAMPLES S1S3 = 184.200 MEAN VALUES S1S3 = 20.467 DISPERSION S1S3 = 0.260 GROUP S2S3 20.2 20.3 20.4 20.4 20.3 20.6 20.6 20.3 20.4 NUMBER OF SAMPLES S2S3 = 9 SUM OF SAMPLES S2S3 = 183.500 MEAN VALUES S2S3 = 20.389 DISPERSION S2S3 = 0.149 GROUP S4S3 20.2 20.3 20.2 20.4 20.4 20.4 20.5 20.2 20.4 NUMBER OF SAMPLES S4S3 = 9 SUM OF SAMPLES S4S3 = 183.000 MEAN VALUES S4S3 = 20.333 DISPERSION S4S3 = 0.100 GROUP S5S3 20.4 20.3 20.4 20.4 20.4 20.5 20.2 20.4 20.2 NUMBER OF SAMPLES S5S3 = 9 SUM OF SAMPLES S5S3 = 183.000 MEAN VALUES S5S3 = 20.356 DISPERSION S5S3 = 0.082 GROUP T2S3 20.4 20.4 20.2 20.2 20.2 20.3 20.2 20.3 20.3 NUMBER OF SAMPLES T2S3 = 9

SUM OF SAMPLES T2S3 = 182.500 MEAN VALUES T2S3 = 20.278 DISPERSION T2S3 = 0.056 GROUP T3S3 20.1 20.4 20.1 20.2 20.2 20.3 20.2 20.1 20.2 NUMBER OF SAMPLES T3S3 = 9 SUM OF SAMPLES T3S3 = 181.800 MEAN VALUES T3S3 = 20.200 DISPERSION T3S3 = 0.080 GROUP T4S3 20.2 20.1 20.2 20.3 20.1 20.4 20.2 20.2 20.1 NUMBER OF SAMPLES T4S3 = 9 SUM OF SAMPLES T4S3 = 181.800 MEAN VALUES T4S3 = 20.200 DISPERSION T4S3 = 0.080 NUMBER OF SAMPLES STANDARD GROUP S3 = 63 SUM OF STANDARD GROUP S3 = 1280 MEAN VALUES OF STANDARD GROUP S3 = 20.31746 STANDARD GROUP S1 19.6 19.3 19.3 19.4 19.6 19.4 19.4 19.3 19.2 NUMBER OF SAMPLES S1 = 9 SUM OF SAMPLES S1 = 174.500 MEAN VALUES S1 = 19.389 DISPERSION S1 = 0.149 CORRECTED MEAN VALUE OF STANDARD GROUP S1 = 19.23968 STANDARD GROUP S2 19.6 19.9 19.9 20.0 19.8 19.9 19.9 20.1 20.0 NUMBER OF SAMPLES S2 = 9

SUM OF SAMPLES S2 = 179.100 MEAN VALUES S2 = 19.9 DISPERSION S2 = 0.160 CORRECTED MEAN VALUE OF STANDARD GROUP S2 = 19.82857 STANDARD GROUP S4 21.2 21.1 21.2 21.1 21.1 21.0 21.2 21.2 21.3 NUMBER OF SAMPLES S4 = 9 SUM OF SAMPLES S4 = 190.400 MEAN VALUES S4 = 21.156 DISPERSION S4 = 0.062 CORRECTED MEAN VALUE OF STANDARD GROUP S4 = 21.13968 STANDARD GROUP S5 21.4 21.5 21.6 21.7 21.8 21.7 21.6 21.8 21.7 NUMBER OF SAMPLES S5 = 9 SUM OF SAMPLES S5 = 194.800 MEAN VALUES S5 = 21.645 DISPERSION S4 = 0.142 CORRECTED MEAN VALUE OF STANDARD GROUP S5 = 21.60635 TEST GROUP T2 19.9 19.8 19.9 19.8 19.8 19.6 19.6 19.9 20.0 NUMBER OF SAMPLES T2 = 9 SUM OF SAMPLES T2 = 178.300 MEAN VALUES T2 = 19.811 DISPERSION T2 = 0.149 CORRECTED MEAN VALUE OF TEST GROUP T2 = 19.85079 TEST GROUP T3 20.3 20.2 20.1 20.1 20.2 20.1 20.2 20.1 20.0 NUMBER OF SAMPLES T3 = 9 SUM OF SAMPLES T3 = 181.300

MEAN VALUES T3 = 20.145 DISPERSION T3 = 0.062 CORRECTED MEAN VALUE OF TEST GROUP T3 = 20.26191 TEST GROUP T4 21.1 21.0 21.1 21.0 21.3 21.2 21.2 21.0 21.2 NUMBER OF SAMPLES T4 = 9 SUM OF SAMPLES T4 = 190.100 MEAN VALUES T4 = 21.122 DISPERSION T4 = 0.096 CORRECTED MEAN VALUE OF TEST GROUP T4 = 21.23968 ENTER VALUE OF STANDARD CONCENTRATION S1 = ? 6.4 ENTER VALUE OF STANDARD CONCENTRATION S2 = ? 8 ENTER VALUE OF STANDARD CONCENTRATION S3 = ? 10 ENTER VALUE OF STANDARD CONCENTRATION S4 = ? 12.5 ENTER VALUE OF STANDARD CONCENTRATION S5 = ? 15.6 PAIRS (X VALUES OF STANDARD CONCENTRATIONS S1-S5, Y CORRECTED MEAN VALUES OF INHIBITION ZONES OF STANDARD GROUPS S1-S5) FOR REGRESSION ANALYSIS X,Y? 6.4, X,Y? 8.0, X,Y? 10, X,Y? 12.5, X,Y? 15.6, 19.23968 19.82857 20.31746 21.13968 21.60635

LOGARITHM FUNCTION WITH A FREE MEMBER Y = A + B * LOG X NUMBER OF DATA POINTS N = 5 SUM OF X = 4.99931 SUM OF X*X = 5.09226 SUM OF Y = 102.13145 SUM OF Y*Y = 2089.85733 SUM OF X*Y = 102.70249

SLOPE (b) = 6.24634 INTERCEPT ON Y AXIS (a) = 14.18088 Y = a + b * X = 14.18088 + 6.24634 * X S.D. (X/Y) = 0.09100 SQUARE ERROR = 0.02484 COEF. OF CORR.= 0.99662 T = 21.00533 D.F.= 3

WOULD YOU LIKE A LIST OF REAL VALUES DIFFERENCES? TYPE YES OR NO @ PRESS RETURN? YES X 6.4 8.0 10.0 12.5 15.6 YOB 19.23968 19.82857 20.31746 21.13968 21.60635 YCALC 19.21665 19.82189 20.42722 21.03255 21.63354 DIFFER 0.02313 6.68619E-03 -0.10976 0.10713 -0.02719

SQUARE ERROR = 0.02484

ASSUMED (DECLARED) POTENCY OF THE SAMPLES: 3300

T2,T3,T4 OBS 19.85079 20.26191 21.23968

S2,S3,S4 OBS 8 10 12.5

S2,S3,S4 CALC 8.08571 9.40880 13. 49182

% 101.07135 94.08803 107.93456

POTENCY OF SAMPLES: 101.03131%, I.E. 3334.03330 On the same manner was done the 2nd test with following results: regression equation parameters: (a) 14.17453, (b) 6.24634., square error (E) 0.02484, coefficient of correlation (R) 0.99662, Student (T) test 21.00533 for 3 degrees of freedom.The biological potency of neomycin in ENBECIN ointment is 3345.85978 I.U./g, i.e. 101.38969% The mean biological potency of neomycin in ENBECIN ointment is 3339.9463 I.U./g., i.e. 101.21050%. Bacitracin (5) The standard solution was prepared in the following concentrations (in I.U./ml): 0.64 (S1), 0.8 (S2), 1.0 (S3), 1.25 (S4) and 1.5 (S5), while the test sample (assumed potency: 500 I.U./g) was prepared in concentrations (in I.U./ml): 0.8 (T2), 1.0 (T3) and

1.25 (T4). Microccocus flavius ATCC 10240 was used as the test organism.Each concentration was tested on 3 agar plates each with 6 holes. Three holes in each Petri plate were filled with the reference standard concentration S3 and the remaining three holes were filled either with one of the standard or sample concentrations. The test was done twice from a single homogenous sample. Monitored parameter: size of diameter of inhibition zones (in mm). FIRST TEST: GROUP S1S3 18.3 18.3 18.3 18.3 18.3 18.4 18.6 18.6 18.7 NUMBER OF SAMPLES S1S3 = 9 SUM OF SAMPLES S1S3 = 165.800 MEAN VALUES S1S3 = 18.422 DISPERSION S1S3 = 0.216 GROUP S2S3 18.1 18.1 18.2 18.4 18.4 18.4 18.4 18.5 18.5 NUMBER OF SAMPLES S2S3 = 9 SUM OF SAMPLES S2S3 = 165.000 MEAN VALUES S2S3 = 18.333 DISPERSION S2S3 = 0.200 GROUP S4S3 18.1 18.1 18.2 18.2 18.3 18.3 18.4 18.5 18.5 NUMBER OF SAMPLES S4S3 = 9 SUM OF SAMPLES S4S3 0 164.600 MEAN VALUES S4S3 = 18.289 DISPERSION S4S3 = 0.189 GROUP S5S3 17.9 18.1 18.2 18.2 18.3 18.3 18.3 18.3 18.4 NUMBER OF SAMPLES S5S3 = 9 SUM OF SAMPLES S5S3 = 164.00 MEAN VALUES S5S3 = 18.222

10

DISPERSION S5S3 = 0.176 GROUP T2S3 18.0 18.1 18.1 18.2 18.2 18.2 18.4 18.4 18.7 NUMBER OF SAMPLES T2S3 = 9 SUM OF SAMPLES T2S3 = 164.300 MEAN VALUES T2S3 = 18.256 DISPERSION T2S3 = 0.362 GROUP T3S3 18.0 18.0 18.1 18.1 18.1 18.4 18.4 18.5 18.6 NUMBER OF SAMPLES T3S3 = 9 SUM OF SAMPLES T4S3 = 164.200 MEAN VALUES T3S3 = 18.245 DISPERSION T3S3 = 0.422 GROUP T4S3 18.1 18.1 18.1 18.3 18.3 18.3 18.4 18.6 18.3 NUMBER OF SAMPLES T4S3 = 9 SUM OF SAMPLES T4S3 = 164.500 MEAN VALUES T4S3 = 18.278 DISPERSION T4S3 = 0.216 NUMBER OF SAMPLES STANDARD GROUP S3 = 63 SUM OF STANDARD GROUP S3 = 1152.4 MEAN VALUES OF STANDARD GROUP S3 = 18.29206 STANDARD GROUP S1 17.1 16.8 16.8 16.9 16.5 16.5 16.5 16.5 16.8 NUMBER OF SAMPLES S1 = 9 SUM OF SAMPLES S1 = 150.400 MEAN VALUES S1 = 16.711 DISPERSION S1 = 0.389

11

CORRECTED MEAN VALUE OF STANDARD GROUP S1 = 16.58095 STANDARD GROUP S2 17.5 17.5 17.6 17.4 17.1 17.3 17.8 17.7 17.7 NUMBER OF SAMPLES S2 = 9 SUM OF SAMPLRES S2 = 157.600 MEAN VALUES S2 = 17.511 DISPERSION S2 = 0.389 CORRECTED MEAN VALUE OF STANDARD GROUP S2 = 17.46984 STANDARD GROUP S4 19.0 19.0 19.1 19.1 18.9 18.9 19.1 19.3 18.9 NUMBER OF SAMPLES S4 = 9 SUM OF SAMPLES S4 = 171.300 MEAN VALUES S4 = 19.333 DISPERSION S4 = 0.140 CORRECTED MEAN VALUE OF STANDARD GROUP S4 = 19.03651 STANDARD GROUP S5 19.6 19.6 19.6 19.7 19.6 19.9 19.9 19.7 19.8 NUMBER OF SAMPLES S5 = 9 SUM OF SAMPLES S5 = 177.400 MEAN VALUES S5 = 19.711 DISPERSION S4 = 0.129 CORRECTED MEAN VALUE OF STANDARD GROUP S5 = 19.78095 TEST GROUP T2 17.7 17.6 17.4 17.4 17.7 17.7 17.6 17.6 17.8 NUMBER OF SAMPLES T2 = 9 SUM OF SAMPLES T2 = 158.500 MEAN VALUES T2 = 17.611 DISPERSION T2 = 0.149 CORRECTED MEAN VALUE OF TEST GROUP T2 = 17.64762

12

TEST GROUP T3 18.4 18.6 18.4 18.1 18.2 18.3 18.5 18.6 18.7 NUMBER OF SAMPLES T3 = 9 SUM OF SAMPLES T3 = 165.800 MEAN VALUES T3 = 18.422 DISPERSION T3 = 0.316 CORRECTED MEAN VALUE OF TEST GROUP T3 = 18.46984 TEST GROUP T4 19.0 19.0 19.1 19.4 19.3 19.3 19.3 19.5 19.3 NUMBER OF SAMPLES T4 = 9 SUM OF SAMPLES T4 = 173.200 MEAN VALUES T4 = 19.245 DISPERSION T4 = 0.242 CORRECTED MEAN VALUE OF TEST GROUP T4 = 19.25873 ENTER VALUE OF STANDARD CONCENTRATION S1 = ? 0.64 ENTER VALUE OF STANDARD CONCENTRATION S2 = ? 0.8 ENTER VALUE OF STANDARD CONCENTRATION S3 = ? 1.0 ENTER VALUE OF STANDARD CONCENTRATION S4 = ? 1.25 ENTER VALUE OF STANDARD CONCENTRATION S5 = ? 1.56 PAIRS (X VALUES OF STANDARD CONCENTRATIONS S1-S5, Y CORRECTED MEAN VALUES OF INHIBITION ZONES OF STANDARD GROUPS S1-S5) FOR REGRESSION ANALYSIS X,Y? 0.64, X,Y? 0.8, X,Y? 1.0, X,Y? 1.25, X,Y? 1.56, 16.58095 17.46984 18.29206 19.03651 19.78095

LOGARITHM FUNCTION WITH A FREE MEMBER Y = A + B * LOG X NUMBER OF DATA POINTS N = 5

13

SUM OF X = -6.95427E-04 SUM OF X*X = 0.09364 SUM OF Y = 91.16032 SUM OF Y*Y = 1668.39764 SUM OF X*Y = 0.75829 SLOPE (b) = 8.23281 INTERCEPT ON Y AXIS (a) = 18.23321 Y = a + b * X = 18.23321 + 8.23281 * X S.D. (X/Y) = 0.05676 SQUARE ERROR = 9.66470E-03 COEF. OF CORR.= 0.99924 T = 44.38744 D.F.= 3

WOULD YOU LIKE A LIST OF REAL VALUES DIFFERENCES? TYPE YES OR NO @ PRESS RETURN? YES X 0.64 0.8 1.0 1.25 1.56 YOB 16.58095 17.46984 18.29206 19.03651 19.78095 YCALC 16.63753 17.43537 18.23321 19.03106 19.82317 DIFFER -0.05657 0.03448 0.05886 5.45736E-03 -0.04222

SQUARE ERROR = 9.66470E-03

ASSUMED (DECLARED) POTENCY OF THE SAMPLES: 500

T2,T3,T4 OBS 17.64762 18.46984 19.25873

S2,S3,S4 OBS 0.8 1.0 1.25

S2,S3,S4 CALC 0.84893 1.06842 1.33219

% 106.11610 106.84215 106.57495

POTENCY OF SAMPLES: 106.51107%, I.E. 532.55534 On the same manner was done the 2nd test with following results: regression equation parameters:(a) 18.32051, (b) 8.23281, square error (E) 0.00967, coefficient of correlation (R) 0.99924, Student (T) test 44.38743 for 3 degrees of freedom. The biological potency of bacitracin in ENBECIN ointment is 521.26329 I.U./g, i.e. 104.25266%.

14

The mean biological potency of bacitracin in ENBECIN ointment is 526.90930 I.U./g., i.e. 105.38186%. ACKNOWLEDGEMENT The authors thanks Mrs. Zlatica Milutinovi for translating the paper into English, and Mrs. Tatjana Josifovi for translating the summary into Italian. REFERENCES 1) Pharmacopoeia Jugoslavica 2000, editio quinta (Ph. Jug. V), Book 1, Biological determinations, 2.7.2. Mikrobiological determinations of antibiotics, Savezni Zavod za zatitu i unapreenje zdravlja, Beograd, 2000, p.101.(Serbian) 2) European Pharmacopoeia (Eur. Ph. 2002), 4th Ed., V. 2.7.2. Microbiological assay of antibiotics, Counsil of Europe, Strasbourg, 2002, p.160. 3) Pharmacopoeia Jugoslavica, editio quatra (Ph. Jug. IV), Vol. I, part 9-102, Examination of penicillin contamination, Savezni Zavod za zdravstvenu zatitu, Beograd, 1984, p.159. (Serbocroatian) 4) USP 26, NF 21: Potencies interpolated from a standard curve, In:<111> Design and analysis of biological assays, United States Pharmacopoeial Convention Inc., Rockville, 2002, p.2033. 5) USP 26, NF 21: < 81> Antibiotics Microbial assays, United States Pharmacopoeial Convention Inc., Rockville, 2002, p.2016.