Assay of Antibiotics by Turbidimetric (or Nephelometric) Methods

ASSAY OF ANTIBIOTICS BY TURBIDIMETRIC (OR NEPHELOMETRIC) METHOD

A large number of antibioics, namely : chlortetracycline, doxycyline, gentamicin, neomycin, streptomycin, tobramycin and the like may be assayed tubidimetrically with fairly good accuracy.

1. Assay of Chlorotetracycline

Theory. Inoculate a medium consisting of : peptone : 6 g, beef extract : 1.5 g, yeast extract : 3 g, sodium chloride : 3.5 g, D-glucose monohydrate : 1.0 g, dipotassium hydrogen orthophosphate : 3.68 g, potassium hydrogen orthophosphate : 1.32 g and dissolve in sufficient water to produce 1 L with a known quantity of a suspension of Staphylococcus aureus (NCTC 6571**) so as to obtain a readily measured opacity after an incubation of about 4 hours. The micro-organisms must exhibit a sensitivity to the antibiotic under investigation to such an extent that a sufficiently large inhibition of growth takes place in the prevailing conditions of the test.

In actual practice, it is always advisable that the inoculated medium should be used immediately after its preparation. Using a phosphate buffer of pH 4.5 (dissolve 13.61 g of KH₂ PO₄ in about 750 ml of water, adjusting the pH to 4.5 with 0.1 M NaOH and diluting to 1 L with water), prepare solutions of the Standard Preparation and the substance under investigation at concentrations presumed to be equal.

To enable the validity of the assay to be examined, it is desirable to use at least three doses of the Standard Preparation and of the substance being examined. It is also advisable to use doses in logarith-mic progression in a parallel line assay.

Materials Required : Standard chlortertracyline ; sterilized media (as described above) : 1 L ; authentic and pure strain of microorganism Staphylococcus aureus (NCTC 6571) ; formaldehyde solu-tion (34–37% w/v) 10 mL ; matched identical test tubes : 20 ;

Procedure : Distribute into identical test-tubes an equal volume of standard tetracycline solution and the sample to be examined (having presumed equal concentrations) and add to each tube an equal volume of inoculated nutrient medium (for instance 1 mL of the solution and 9 ml of the medium). Prepare at the same time two control tubes without the chlorotetracycline, one containing the inoculated medium and the other identical with it but treated immediately with 0.5 mL of formaldehyde solution. These tubes are used to set the optical apparatus employed to measure the growth.

Place all the tubes, randomly distributed, in a water-bath or other suitable means of bringing all the tubes rapidly to 35–37°C i.e., the incubation temperature and maintain them at that temperature for 3 to 4 hours, taking due precautions to ensure uniformity of temperatures and identical incubation times. After incubation, stop the growth of the microorganisms by adding 0.5 mL of formaldehyde solution, each tube and subsequently measures the opacity to at least three significant figures using a suitable optical apparatus. From the results calculate the potency of the substance being examined i.e., chlortetracycline by standard statistical methods.*

Note. (a) Rectilinearity** of the dose-response relationship, transformed or untransformed, is often obtained only over a very limited range. It is this range that must be used in calculating the activity and it must include at least three consecutive doses in order to permit rectilinearity to be verified,

(b) Use in each assay the number of replications per dose sufficient to ensure the required precision. The assay may be repeated and the results combined statistically to obtain the required precision and to ascertain whether the potency of the antibiotic being examined is not less than the minimum required.

2. Cognate Assays

A few other official antibiotics in BP (1993) may also be assayed by adopting the method stated above, but using specific micro-organism, definite final pH of the medium, pH of the phosphate buffer, potency of solution (U per ml) an the incubation temperature. A few typical examples are given in Table 10.6 below :

Table 10.6. Assay of Antibiotics Turbidimetrically

3. Assay of Vitamins

As or late the judicious exploitation of various microorganisms as dependable and reliable ‘analystical tools’ in a well organized Quality Assurance Laboratory (QAL) for the precise determi-nation of a plethora of Vitamins and amino acids.

Merit of Microbial Assays. There are several well-known merits of microbial assays as enu-merated under :

(1) These are as precise and accurate as the ‘chemical methods’.

(2) These are invariably quite simple, convenient, not-so-cumbersome, and above all definitely inexpensive.

(3) A very small quantum of the ‘sample’ is required for the recommended microbial assay.

(4) They hardly need any elaborated instrumentation.

(5) These microbial assays do require the following essential criteria, such as :

·        ascertains continuous checks for consistency of results,

·        ensures specificity, and

·        prevents any possible interferences.

(6) Automation of microbial assays may essentially overcome any possible limitations, accu-racy of observations, and the sample-handling capacity to a significant extent.

Example : In an ‘automatic photometric assay’ the following activities do take place in a sequential manner, namely :

·        measures the exact quantum of antibiotic present in a given solution,

·        incorporates requisite quantum of inoculum and nutrient medium,

·        incubates the resulting mixture for 100 minutes,

·        transfers the incubated mixture to photometer cell, and

·        results are adequately read and recorded.

Principle. It has been amply proved and established that there are some specific microbes which predominantly require vitamin (factor) for their usual normal growth phenomenon ; and, therefore, are quite sensitive to the extremely small quantities of the much desired ‘factor’. Nevertheless, it is pre-cisely the critical inherent ability of these particular microorganisms (i.e., the ‘test organisms’) to carry out the synthesis of the ‘factor’ being determined. This ultimately gives rise to the fundamental basis of the microbial assay of vitamins. To accomplish the ultimate objective, the ‘test organism’ is duly inoculated in the highly specialized culture media that are essentially complete in every possible re-spects except the presence of the ‘factor’ under investigative study. In reality, it evidently caters for the ‘control’ wherein either little or almost minimal growth of microbes is duly exhibited. Importantly, in another set of parallel/identical experiments, one may incorporate meticulously the ‘graded quantities of factor’ the thus the ultimate growth of the test organism (i.e., response) is observed adequately. However, one may observe invariably that the ‘response’ (i.e., growth of the ‘test organism’) is directly proportional to the ‘factor’ (i.e.,quantum of the dose) actually incorporated to the culture medium.

Microbial assays of the following three water-soluble vitamins would be discussed individually in the sections that follows :

(1) Calcium Pantothenate,

(2) Niacin (or Niacinamide), and

(3) Vitamin B₁₂ (or Cyanocobalamin).

(1) Calcium Pantothenate

It refers to one of the B complex vitamins (or vitamin B complex). The various steps involved for the assay are enumerated under sequentially :

(1) Reagents. The various reagents essentially required for the assay of ‘calcium pantothenate’ are :

(a) Standardized Stock Solution. Each mL of this stock solution consists of 50 mcg of cal-cium panthothenate. It may be prepared by carefully dissolving 50 mg of BPCRS* calcium pantothenate in 500 mL of double-distilled water ; 10 mL of 0.2 M acetic acid, 100 mL of 1.6% (w/v) sodium acetate ; and volume made upto 1 L with DW.

Note : The resulting solution must be stored under a layer of ‘toluene’ in a refrigerator.

(b) Standard Solution. The standard solution should contain approximately 0.04 mcg of cal-cium pantothenate in 1 mL, and is duly prepared by diluting the Standard Stock Solution (a).

(c) Test Solution. The test solution essentially contains nearly the same equivalent amount of calcium pantothenate as present in the Standard Solution (a) above i.e., 0.4 mcg.mL^–1 prepared in double-distilled water.

(d) Culture Medium. The culture medium is composed of the following solutions and ingredients :

Casein hydrolysate solution** : 25 mL

Cysteine-tryptophane solution : 25 mL

Polysorbate-80 solution*** : 0.25 mL

Dextrose (anhydrous) : 10 g

Sodium acetate (anhydrous) : 5 g

Adenine-guanine-uracil solution : 5 mL

Riboflavin-Thiamine hydrochloride-Biotin Solution : 5 mL

PABA****-Niacin-Pyridoxine hydrochloride solution : 5 mL

Calcium pantothenate solution A : 5 mL

Calcium pantothenate solution B : 5 mL

The culture medium is usually prepared by dissolving both anhydrous dextrose and sodium acetate in previously mixed solutions and the pH is carefully adjusted to 6.8 with 1 M.NaOH solution. The final volume is duly made upto 250 mL with distilled water and mixed thoroughly.

(2) Stock Culture of Organism : The stock culture of organism may be prepared dissolving 2 g water-soluble yeast extract in 100 mL DW, 500 mg anhydrous dextrose, 500 mg anhydrous sodium acetate, and 1.5 g agar. The resulting mixture is heated gently so as to dissolve the agar. Now, 10 mL of hot solution is transferred to test tubes and sterilized at 121°C by keeping in an upright position. The ‘stab culture’***** is now prepared duly in three tubes employing Lactobacillus plantarum, incubated at 30 to 37°C for 16 to 24 hours, and stored in a refrigerator ultimately.

(3) Preparation of Inoculum. The cells consequently obtained from the stock culture, (a) above, organism are duly transferred to a sterile tube containing 10 mL of the culture emdium (d). Finally, it is incubated at 30 to 37°C for a duration of 16–24 hours.

(4) Methodology. The various steps involved are as stated below :

(i) Standard Solution (b) is added to five test tubes in varying amounts viz., 1, 2, 3, 4 and 5 mL in duplicate.

(ii) To each of the five above test tubes plus another four similar tubes without any standard solution is added 5 mL of culture medium, and the final volume made upto 10 mL with DW.

(iii) Now, volumes of test solution (c) corresponding to either three or more of the levels as taken above, are incorporated carefully to similar test tubes, in duplicate.

(iv) To each test tube 5 mL of the medium solution, and volume is made upto 10 mL with DW. Thus, we may have two separate racks :

First Rack : Having complete set of standard plus assay tubes ; and

Second Rack : Having duplicate set only.

(v) Tubes of both the series are duly heated in an autoclave at 121°C for 5 minutes only ; cooled to ambient temperature, added 1 drop of inoculum (3) to each tube except two of the four tubes that specifically has no ‘standard solution’ (i.e., the uninoculated tubes), and mixed thoroughly. The tubes are adequately incubated at 121°C at 30–37°C for 16– 24 hours.

(vi) Transmittance of the various tubes is measured with a spectrophotometer at wave-length ranging between 540–660 nm.

(5) Calculation. First of all, a standard concentration response curve is plotted between the transmittance Vs log mL (volume) of the standard solution in each tube. In this way, the response is duly calculated by summing up the two transmittances for each level of the test solution.

Finally, the exact concentration of the calcium pantothenate in the ‘test sample’ is determined accurately with the aid of the standard concentration-response curve obtained.

(2) Niacin (or Niacinamide)

Preamble. In this particular assay the most appropriate organism should be such that must be able to fully use up there five vital and important components, namely : niacin, nicotinuric acid, miacinamide, niacinamide, nucleoside, and coenzymase (an enzyme). This organism that may criti-cally satisfy the aforesaid requirements happens to be Lactobacillus plantarum. Interestingly, this acid forming organism is found to be quite incapable to afford the synthesis of niacin for its on-going meta-bolic processes. A few other equally important criteria of this organism are as given under :

·        Non-pathogenic in nature

·        Easy to culture

·        Least affected by various stimulatory or inhibitory constituents usually present in ‘pharma-ceutical formulations’ containing niacin.

·        Conveniently grown upon a rather simple stab culture comprising of gelatin, yeast extract, and glucose.

Note. (1) For the assay of niacin, it is cultured in the assay tubes by actually transferring to the ensuing liquid culture medium comprising of the basic medium having an optimized quan-tum of added niacin.

(2) To obtain a measurable response the amount of niacin present in each tube may range between 0.05 to 0.5 mcg.

(1) Reagents. The various reagents used for the microbial assay of niacin are as enumerated under :

(a) Standard Stock Solution of Niacin (I). It essentially contains 100 mcg.mL^–1 of niacin USPCRS*.

(b) Standard Stock Solution of Niacin (II). It consists of 10 mcg.mL^–1 of niacin USPCRS; and is prepared by dilution of solution (I) in the ratio 1 : 10, i.e., 1 mL of solution (I) is made up to 10 mL in DW.

(c) Standard Niacin Solution. It critically contains niacin ranging between 10-40 ng (i.e.,nanogram). mL^–1, and may be prepared from Solution II by an appropriate dilution with DW.

(d) Basal Culture Medium Stock Solution. The basal culture medium stock solution may be prepared by the following requisite proportion of various ingredients and solutions as enu-merated under :

Casein hydrolysate solution** : 25 mL

Cystine-tryptophane solution : 25 mL

Anhydrous dextrose : 10 g

Anhydrous sodium acetate : 5 g

Adenine-guanine-uracil solution : 5 mL

Riboflavin-Thiamine hydrochloride-Biotin Solution : 5 mL

PABA-Calcium patothenate-Pyridoxine

hydrochloride solution : 5 mL

Niacin solution A : 5 mL

Niacin solution B : 5 mL

The culture medium is duly perpared by carefully dissolving anhydrous dextrose and anhydrous sodium acetate into the previously mixed solutions, and adjusting the pH precisely to 6.8 by the dropwise addition of 1 M.NaOH. The final volume was made up to 250 mL with DW.

(e) Culture Medium. Into a series of labeled ‘test tubes’ containing 5 mL of the Basal Culture Medium Stock Solution [(d) above] 5 mL of water containing exactly 1 mcg of niacin are incorporated carefully. The sterilization of all these ‘test tube’ are carried out by first plug-ging each of them with cotton, and subsequently autoclaving them at 121°C for 15 minutes.

(2) Preparation of Inoculum. Transfer from the stock culture of Lactobacillus plantarum cells aseptically into a sterilie test tube containing 10 mL of culture medium [(e) above]. The resulting culture is duly incubated at a temperature ranging between 30–37°C for a duration of 16–24 hours. The cell suspension of the said organism is termed as the inoculum.

(3) Methodology. The various steps that are involved in the microbial assay of niacin are de-scribed as under in a sequential manner :

(i) First and foremost the ‘spectrophotometer’ is duly calibrated according to the proce-dural details mentioned in the ‘official compendia’*.

(ii) Standard Niacin Solution is added in duplicate into various Standard Niacin Tubes in varying quantities viz., 0, 0.5, 1.0, 1.5, 2.0, 2.5 ...... 5.0 mL respectively. To each of these tubes add 5.0 mL of the Basal Culture Medium Stock Solution [(d) above] plus sufficient distilled water to make 10 mL.

(iii) Test Solution Tubes containing varying amounts of niacin are carefully prepared by making in duplicate 1, 2, 3, 4, and 5 mL respectively of the ‘test solution’. To these tubes are added 5 mL of the Basal Culture Medium Stock Solution [(d) above], and followed by water to make upto 10 mL.

(iv) All the tubes obtained in (iii) above are duly plugged with cotton, and adequately steri-lized in an ‘autoclave’ (for 15 minutes at 121°C).

(v) After having brought down the hot tubes to the ambient temperature, they are carefully inoculated asepticlly with one drop of inoculum [(2) above], and subsequently be-tween 30–37°C for a duration of 16 to 24 hours.

(vi) Having set the percentage transmittance at 1 for the ‘uninoculated blank’, the various transmittance of the inoculated tubes is duly noted, and recorded.

(4) Calculation : First of all a ‘Standard Curve’ is plotted for niacin between :

·        standard transmittances for each level of Standard Niacin Solution, and

·        exact quantum of niacin (in mcg) present duly in the respective tubes.

Thus, from the ‘Standard Curve’, one may easily obtain the niacin precisely present in the ‘test solution’ of each tube by interpolation.

Finally, the exact niacin content of the ‘test material’ may be calculated from the ‘average values’ duly obtained from at least six tubes which should not vary by more than ± 10% with respect to the average values.

(3) Vitamin B₁₂ [or Cynocobalamin]

It is pertinent to state here that the ‘basic culture medium’ employed for the assay of vitamin B₁₂ is found to be extremely complex in nature, and essentially comprises of a large number of varying constituents in the form of a mixture in solution.

Various steps are as follows :

(1) First set of tubes contains solely the measured quantum of a Standard Cyanocobalamin Solution.

(2) Second set of tubes essentially comprise of the graded volumes of the ‘test sample’ (i.e., unknown).

(3) All the ‘tubes’ (i.e., first set + second set) are carefully inoculated with a small quantity of the culture of Lactobacillus leichmanni, and subsequently incubated duly.

(4) The precise extent of growth is assayed by measuring the percentage transmittance by the help of a standardized (calibrated) spectrophotometer.

(5) The concentration-response curve is now prepared mediculously by plotting the following two observed parameters :

·        Transmittance values (i.e., response), and

·        Different concentrations (i.e.,dose) of Standard cyanocobalamin solution.

(6) Ultimately, the exact quantum of vitamin B₁₂ duly present in the given ‘test sample’ (i.e., unknown) is calculated based on the ‘Standard Curve’ by the interpolation.

4. Assay of Amino Acids

As discussed earlier the critical and specific requirements of a microorganism for an ‘amino acid’ may be employed categorically to assay the exact quantum of the amino acid duly present in a plethora of pharmaceutical formulations or even food products by allowing the particular organism to grow optimally in a medium containing all the ‘essential requirements’, and thus the measured doses of the ‘substance’ called be assayed accurately.