INTRODUCTION:

Malnutrition refers to the poor diet or lack of food. It results when the intake of nutrients or energy is too high, too low, or poorly balanced. Undernutrition can lead to delayed growth or wasting, while a diet that provides too much food, but not necessarily balanced, leads to obesity. Malnutrition involves a dietary deficiency¹. People may eat too much of the wrong type of food and have malnutrition, but this article will focus on under nutrition, when a person lacks nutrients because they do not consume enough food².

Parenteral nutritional formulations:

Parenteral Nutritional (PN) formulations are used to feed a person intravenously using nutritional formulae containing essential nutrients such as electrolytes, glucose, amino acids and vitamins³. These are customized form of standardized formulations or prepared as per individual patients need in hospital pharmacy or institution. During the preparation, there are high chances of preparation errors if procedure is not followed carefully or maybe because of prescribing error⁴.

Inductively coupled plasma:

The main analytical advantage of the ICP over the other excitation sources originate from its capability for efficient and reproducible vaporization, atomization, excitation and ionization for a wide range of elements in various sample matrices. This is possible mainly due to the high temperature, 7000-10,000 K in the observation zone. This temperature is much higher than the maximum temperature of flames and furnace in FES (flame emission spectroscopy) and AAS (Atomic Absorption Spectroscopy)⁵.

Principle involved in ICP-OES technique:

ICP-OES is a powerful analytical tool for the determination of trace elements. In this technique, photons are emitted from the excited atoms and ions when they are passed through plasma. Solid, liquid and gas samples can be analyzed by this technique. Liquid samples may be injected directly into the instrument⁶. The sample solution is converted to an aerosol and directed into the middle part of the plasma. At its core the inductively coupled plasma sustains a temperature of approximately 10,000 K, so the aerosol is quickly vaporized. Aerosol particles are liberated as free atoms in the gaseous state⁷. Further excitation in the plasma imparts additional energy to the atoms, promoting them to excited states. As plenty of energy available in plasma atoms are converted into the ions and ions are further excited. Both the atomic and ionic excited state species then relax to the ground state via emission of a photon⁸. These photons have characteristic energies that are determined by the quantized energy level structure for the atoms or ions. This characteristic wavelength of the photons can be used to identify the elements from which they originated. The total number of photons is directly proportional to the concentration of the originating element in the sample^9,10.

The present study aims to formulate the nutritional formulation (that is used to combat malnutrition) containing various amino acids and evaluate it by using ICP-OES technique to combat malnutrition.

MATERIALS AND METHODS:

Apparatus and Instruments:

Class A glass and plastic volumetric flasks of appropriate volumes, plastic bottle of appropriate volumes, disposable polypropylene tubes, disposable plastic tips of appropriate size for automatic pipette, weighing balance (Mettler Toledo XP 011), ultrasonicator (Ana matrix), water Purifier (Milli-Q, Merck Millipore), automatic pipette (Eppendrof), cooling chamber (New Tronic), ICP-OES system (Perkin Elmer Optical Emission Spectrophotometer Optima 7300V (software: Winlab32)

Chemicals and reagents:

All the amino acids and nitric acid were procured from Sigma Aldrich, L-phenylalanine was procured from Cal biochem, and yttrium standard 1000mg/L was obtained from In-organic ventures.

Methods:

Preparation of Nutritional formulation:

All the amino acids like L-Alanine, L-Arginine, L-Aspartic acid, L-Cysteine, L-Glutamic acid, Glycine, L-Histidine, L-Isoleucine, L-Leucine, L-Lysine Monohydrate, L-Methionine, L-Ornithine-hydro-chloride, L-Phenylalanine, proline, L-Serine, L-Taurine, L-Threonine, L-Tryptophan, L-Tyrosine, L-Valine of required quantity were weighed and were transferred into a 1000mL of volumetric flask, and then the required quantity of electrolytes like potassium acetate, calcium chloride di-hydrate, magnesium acetate tetra hydrate were added and finally made up the volume with Milli-Q Water¹¹.

Preparation of various solutions:

1. Rinse solution preparation:

Rinse solution for plasma flushing, during and before the analysis was used, to avoid carry over of elements on the torch. The rinse solution was prepared by using 100ml of 67% w/v of nitric acid and made up the volume to 2000ml with milli-Q-water¹².

2. Internal Standard Solution (IS):

Internal standard solution was prepared by adding 5mL of yttrium stock standard of 1000mg/L and 20ml of nitric acid into a 100ml volumetric flask and made up the volume with the milli-Q-Water¹³.

3. Magnesium standard stock solution (Mg-Stock):

Magnesium standard solution was prepared by pipetting 5mL of 1000mg/L Magnesium standard stock solution into a 100ml volumetric flask and made up the volume with the milli-Q-water.

4. Preparation of Magnesium intermediate solution:

Magnesium intermediate solution was prepared by pipetting 2.5ml of 50mg/L of magnesium standard solution into a 25ml of volumetric flask and the volume was made upto mark with the milli-Q-water.

5. Working Standard solution (WS):

Working standard was prepared as per the calibration curve range for working standard (80%-110%) by volumetrically transferring the required volume of elemental standard, ISS solution and diluted to required volume with purified water. Preparation of test Solution was done by using Na standard solution of 0.5, 0.7, 0.9, 1.1, 1.3ml from 1000mg/L for WS-1, WS-2, WS-3, WS-4, and WS-5, K Standard solution of 1.1, 1.3, 1.5, 1.8, 2.2 ml from 1000mg/L and Ca Standard solution of 0.5, 0.7, 0.9, 1.1, 1.3 from 1000mg/L and then add Mg stock 50mg/L of 0.8, 1, 1.4, 1.8, 2.2 then the internal solution was added for all the WS-1, WS-2, WS-3, and WS-4 and WS-5 then it was finally made with Milli-Q-Water.

6. Control standard preparation (CS):

Control standard solution which was used to monitor the performance of instrument. To assess the performance for the test samples, control standard was prepared same as that of the standard calibrating working standard solution, by spiking elemental standard, matrix solutions and internal standard stock solution and final volume made up with the milli-Q-water.

7. Preparation of validation samples (VS):

Preparation of validation samples was done by using Na standard solution of 0.7, 0.8, 0.9, 1.0, 1.1ml from 1000 mg/L for VS-1 ,VS-2, VS-3, and VS-4 and VS-5, K Standard solution of 1.1, 1.3, 1.5, 1.7, 1.9ml from 1000 mg/L and Ca Standard solution of 0.7, 0.8, 0.9,1.0,1.1ml from 1000mg/L and then add Mg IS (mL) of 1.0, 1.2, 1.4, 1.6, 1.8ml then the internal solution was added for all the TS-1 ,TS-2,TS-3, and TS-4 of 1, 1, 1, 1, and then it was finaly made up with the Milli-Q-Water.

8. Accuracy:

The accuracy of an analytical procedure expresses the closeness of agreement between the value which is accepted either as a conventional true value or an accepted reference value and the value found. This is sometimes termed trueness. Accuracy was determined for three replicates like R1, R2, R3 for three sets of validation samples (VS1, VS2, VS3)¹⁴.

9. Precision:

The precision of an analytical procedure expresses the closeness of agreement (degree of scatter) between a series of measurements obtained from multiple sampling of the same homogeneous sample under the prescribed conditions.

10. Linearity:

The calibration curve for sodium, potassium, calcium and magnesium is determined by plotting the theoretical value mg/L, against experimental concentration mg/L.

11. Specificity:

Specificity is the ability to assess unequivocally the analyte in the presence of components which may be expected to be present. Typically, these might include impurities, degradants, matrix, etc. Lack of specificity of an individual analytical procedure may be compensated by other supporting analytical procedure(s).

12. Robustness:

The robustness of an analytical procedure is a measure of its capacity to remain unaffected by small, but deliberate variations in method parameters and provides an indication of its reliability during normal usage. Robustness was performed at two condition by varying the nebulizer gas flow at nominal condition at 0.82 L/min and at the varied conditions of 0.84 L/min.

13. System suitability criteria:

System suitability criteria for all the elements like Na, k, Ca and Mg was calculated based on the working standards linearity of WS-1, WS-2, WS-3, WS-4, WS-5, measurement precision expect for the blank the relative standard deviation is said to be < 1.5% for all the standards, and the control standard accuracy recovery is calculated.

RESULTS AND DISCUSSIONS:

Accuracy:

Based on the data obtained for Na, K, Ca and Mg respectively was found to be concurrent to its theoretical value and hence, the results conclude that the obtained values for Na (98.2-99.1%), K (99.0-99.7%), Ca (98.5-99.7) and Mg(97.6-98.1) were found to be accurate.

Precision:

Based on the data obtained it was found that two injections of same solution does not vary and concentration obtained for two injections are precise, the percentage difference between the two injection were within the limits (1.5%).

Linearity:

The linearity of all the elements was found to be within the limit (1.000), the co-relation coefficient between the experimental and theoretical value was found to meet its acceptance criteria hence the values were found to be a valid results. The results are depicted in table 1 and 2 for sodium, potassium, calcium, magnesium respectively with graphical representation in figure 01 as shown below.

Table 1: Linearity of sodium and potassium

Sodium				Potassium
Replicates	Theoretical Concentration mg/L	Experimental Concentration mg/L	% Mean recovery	Theoretical Concentration mg/L	Experimental Concentration mg/L	% Mean recovery
TS-1	7.04	7.82	111.0	11.0	12.11	110.1
TS-2	8.05	8.43	104.7	13.0	13.48	103.7
TS-4	9.06	9.45	104.0	15.0	15.44	103.0
TS-5	10.07	10.60	105.3	17.0	17.65	104.1
TS-6	11.07	11.50	103.9	19.0	19.60	103.2

Table 2: Linearity of calcium and magnesium

Calcium				Magnesium
Replicates	Theoretical Concentration mg/L	Experimental Concentration mg/L	% Mean recovery	Theoretical Concentration mg/L	Experimental Concentration mg/L	% Mean recovery
TS-1	6.99	7.38	105.6	0.5	0.58	117.3
TS-2	7.99	7.96	99.7	0.6	0.64	108.0
TS-4	8.99	8.97	99.8	0.7	0.75	107.2
TS-5	9.99	10.13	101.4	0.8	0.86	108.4
TS-6	10.98	11.01	100.2	0.9	0.96	106.9

Fig 01: Linearity graph of Na, K, Ca and Mg

Specificity:

Blank formulation showed negative response in the absence of analytes, formulation containing analytes showed the positive response for Na, K, Ca and Mg.

There was no interference found with the nutritional formulation, hence the product was said to be specific.

Robustness:

Robustness was carried out by varying the nebulizer flow rate at the nominal condition at 0.82 L/min and varied condition at 0.84 L/min, the results concluded that these variations does not have any impact on the results.

System suitability criteria:

All the system suitability results met the acceptance criteria, like control standard accuracy (98-102%), sample precision (<1.5%), measurement precision except for the blank. All the working standards met their acceptance criteria of relative standard deviation (<1.5%), and thus co-relation co-efficient value was found to be (0.9995) thus it passed all the System suitability criteria’s.

Range:

Validated range for Na, k, Ca, and Mg was found to be 911.95mg/L, 1525.6mg/L, 940.54mg/L, 71.402mg/L respectively. In this range the product was said to be accurate (98-102), precision (1.5), and linearity (1.000) meets the acceptance criteria.

CONCLUSION:

The formulated nutritional product was considered to be within the specific range, also the method validation studies was performed for Na, k, Ca and Mg present in nutritional formulation by using ICP-OES instrument, and was found to be cost effective.

It is a simple fast, and reliable method for ICP-OES determination of Na, k, Ca, and Mg in the nutritional formulation. The data generated for the determination of Na, k, Ca and Mg was considered to be a valid results as per ICH Q2 R1 guidelines with respect to linearity, accuracy, specificity and robustness at varied conditions.

Finally it can be concluded that the nutritional formulation has a great scope in the field of human health care and can be utilized for achieving the food and nutritional fulfillment of the nation to combat malnutrition

ACKNOWLEDGEMENT:

The authors are thankful to dean and management of College of Pharmaceutical Sciences, Dayananda Sagar University, Bengaluru, for their constant support. We also thank Syngene International Private Limited, Bengaluru, for supporting us to carryout the current research work.

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Received on 04.09.2019 Modified on 11.11.2019

Research J. Pharm. and Tech. 2020; 13(8):3733-3737.

DOI: 10.5958/0974-360X.2020.00661.7