Application Note No. 230/2016

Transcription

1 Application Note No. 230/2016 Nitrogen determination in bovine serum albumin KjelDigester K-436, KjelFlex K-360: Comparison of standard-kjeldahl and micro-kjeldahl for nitrogen determination in pharmaceutical products.buchi.com Quality in your hands

2 1. Introduction A simple and fast procedure for nitrogen determination in bovine serum albumin, a protein applied in many biochemical and medical applications, according to AOAC and standard of Chinese pharmacopeia is introduced [1,2]. For many pharmaceutical products, the nitrogen content is determined to evaluate the production process and the product quality. Therefore, the sample is digested with sulfuric acid and the Kjeldahl tablet using the SpeedDigester K-436 followed by standard- and micro- Kjeldahl methods. The micro-kjeldahl method is applied in order to save valuable resources for samples with a high nitrogen content. The nitrogen content in bovine serum albumin determined by standard- and micro-kjeldahl methods was compared. 2. Equipment SpeedDigester K-436 (the parameters used are also valid for K-425) Scrubber K-415 TripleScrub ECO KjelFlex K-360 with titration set 877 Titrino plus (Metrohm) Sample tubes micro, BUCHI ( ) Insulation plate micro, BUCHI ( ) Analytical balance (accuracy ± 0.1 mg) 3. Chemicals and Materials Chemicals: Sulfuric acid, conc. 98 %, analytical reagent, Beijing Chemical Works Titanium Micro, BUCHI Kjeldahl Tablets ( ) Titanium, BUCHI Kjeldahl Tablet ( ) Sodium hydroxide 32 %, analytical reagent, Sinopharm Chemical Reagent Boric acid 2 %, 100 g boric acid (analytical reagent, Tianjin Guangfu Fine Chemical Research Institute) diluted to 5 L with deionized water, adjust ph to 4.65 Sulfuric acid 0.05 mol/l, Merck ( ) Sulfuric acid 0.01 mol/l, Aladdin (S L) Neutralization solution for the Scrubber: 600 g sodium carbonate (analytical reagent, Sinopharm Chemical Reagent) about 2 ml ethanol and a spatula tip of bromthymol blue (analytical reagent, Tianjin Kemiou Chemical Reagent) diluted to 3 L with distilled water Glycine, assay 99.5 %, Sinoharm Chemical Reagent For a safe handling please pay attention to all corresponding MSDS. Samples: Bovine serum albumin: Sample, nitrogen content about 1.6 mg/ml and 10 % glycerin. The samples was provided from TanjinCanSino Biotechnology Inc, China. 4. Procedure The determination of nitrogen and protein in pharmaceutical products includes the following steps: Application Note 230/2016 June /7

3 Digestion of the sample, using the K-436 (K-425 respectively) Distillation and titration of the sample, using KjelFlex K Digestion of glycine with the standard-kjeldahl method (verification of the method) Start the SpeedDigester K-436 according to the parameters listed in Table 1 1. Preheat the SpeedDigester K-436 at level 5 for 10 min. 2. Place 0.05 g glycine in a 300 ml sample tube 3. Add one Titanium tablet and 10 ml of sulfuric acid (conc. 98 %) to each tube 4. Prepare additional blanks, chemicals without sample 5. Connect the Scrubber K-415 to the K-436 for absorbing the acid fumes created during 6. Insert the rack into position of the SpeedDigester and immediately start the according to the parameters listed in Table 1 7. Let the samples cool down when the is completed Table 1: Temperature profile for with the K-436. Step Heating level Time [min] Cooling - 30 Digestion time Digestion of glycine with the Mirco-Kjeldahl method (verification of the method) Start the SpeedDigester K-436 according to the parameters listed in Table 2 1. Install the insulation plate for micro sample tubes on the SpeedDigester 2. Preheat the SpeedDigester K-436 at level 5 for 10 min 3. Place 0.05 g glycine in a 100 ml sample tube 4. Add one Titanium Micro tablet and 3 ml of sulfuric acid (conc. 98 %) to each tube 5. Prepare additional blanks, chemicals without sample 6. Connect the Scrubber K-415 to the K-436 for absorbing the acid fumes created during 7. Insert the rack into position of the SpeedDigester and immediately start the according to the parameters listed in Table 2 8. Let the samples cool down when the is completed Table 2: Temperature profile for with the K-436. Step Heating level Time [min] Cooling - 30 Digestion time Digestion of samples analyzed according to the standard-kjeldahl method 1. Place 1 ml sample in a 300 ml sample tube 2. Start the SpeedDigester K-436 according to the parameters listed in Table 1 3. Add one Titanium tablet and 10 ml of sulfuric acid (conc. 98 %) to each tube 4. Prepare additional two blanks, i.e. add 10 ml sulfuric acid and one Titanium tablet without sample 5. Connect the Scrubber K-415 to the K-436 for absorbing acid fumes created during 6. Insert the rack into the position of the KjelDigester and immediately start the according to the parameters listed in Table 1 7. Let the samples cool down when the is completed NOTE: If the liquid inside the sample tube is not clear and green, digest for additional 15 min at Level 9. The samples should be clear-green immediately after the. A darkening Application Note 230/2016 June /7

4 of the clear liquid samples during the cooling down process is possible but does not affect the results Digestion of samples analyzed according to the Mirco-Kjeldahl method 1. Place 0.5 ml sample in a 100 ml sample tube 2. Install the insulation plate for micro sample tubes on the SpeedDigester 3. Start the SpeedDigester K-436 according to the parameters listed in Table 2 4. Add one Titanium Micro tablet and 3 ml of sulfuric acid (conc. 98 %) to each tube 5. Prepare additional blanks, i.e. add 3 ml sulfuric acid and one Titanium Micro tablet without sample 6. Connect the Scrubber K-415 to the K-436 for absorbing acid fumes created during 7. Insert the rack into the position of the KjelDigester and immediately start the according to the parameters listed in Table 2 8. Let the samples cool down when the is completed NOTE: If the liquid inside the sample tube is not clear and green, digest for additional 15 min at Level 9. The samples should be clear-green immediately after the. A darkening of the clear liquid samples during the cooling down process is possible but does not affect the results Distillation and titration The parameters set on the KjelFlex K-360 for micro-kjeldahl and the standard-kjeldahl are listed in Table 3 and Table 4 respectively. Table 3: Distillation and titration parameters for the KjelFlex K-360 and titration applying mirco-kjeldahl. KjelFlex K-360 Metrohm 877 Titrino plus H 2O volume 25 ml Titration solution H 2SO mol/l NaOH volume 30 ml Sensor type ph electrode H 3BO 3 50 ml Measuring mode Endpoint ph=4.65 Reaction time 5 s Titration rate Optimal Steam Power 100 % Stop Criterion Drift Distillation time 240 s Stop Drift 20 µl/min Tiration Start 240 s Stop Time Off Titration type Boric acid Filling Rate max. ml/min Stirrer speed distillation 5 Stirrer speed titration 7 Table 4: Distillation and titration parameters for the KjelFlex K-360 and titration applying standard-kjeldahl KjelFlex K-360 Metrohm 877 Titrino plus H 2O volume 50 ml Titration solution H 2SO mol/l H 2SO mol/l (for the glycine standard Kjeldahl) NaOH volume 60 ml Sensor type ph electrode H 3BO 3 50 ml Measuring mode Endpoint ph=4.65 Reaction time 5 s Titration rate Optimal Steam Power 100 % Stop Criterion Drift Distillation time 240 s Stop Drift 20 µl/min Tiration Start 240 s Stop Time Off Titration type Boric acid Filling Rate max. ml/min Stirrer speed distillation 5 Stirrer speed titration 7 Application Note 230/2016 June /7

5 4.6. Calculation The results are calculated as a percentage of nitrogen. The following equations (1) are used to calculate the results. For the reference substance, the purity of the glycine is considered in equation (2), the reconery results of the glycine is considered in equation (3). w N (V = Titrant - V V Blank Sample ) z c f M N (1) %N 100 %N Gly = (2) P N actual Recovery = *100 (3) N theoretical w N : nitrogen content [mg/ml] V Titrant : amount of titrant for the sample [ml] V Blank : mean amount of titrant for the blank [ml] z : molar valence factor (1 for HCl, 2 for H 2 SO 4 ) c : titrant concentration [mol/l] f : titrant factor (for commercial solutions normally 1.000) M N : molecular weight of nitrogen ( g/mol) V Sample : sample volume [ml] %N : percentage of weight of nitrogen %N Gly : percentage of weight of nitrogen corrected for the purity of reference substance glycine [%] P : percentage of weight of protein Recovery: the recovery result of glycine [%] 5. Results 5.1. Recovery of glycine according to the standard-kjeldahl method The three results of nitrogen determination and recovery for glycine analysis (assay 99.5 %) are presented in Table 5. The nominal value of glycine is % nitrogen. The recoveries are between 99.8 % and %. Table 5: Results of the recovery of nitrogen in glycine applying standard-kjeldahl (using 0.05 mol/l H 2SO 4). Glycine m Sample [g] V Sample [ml] %N Gly Recovery [%] Sample Sample Sample Average [%] Rsd [%] The mean blank volume (V Blank ) was ml (n = 2) Recovery of glycine according to the Mirco-Kjeldahl method The four results of nitrogen determination and recovery for glycine analysis (assay 99.5 %) are presented in Table 6. The nominal value of glycine is % nitrogen. The recoveries are between % and %. Application Note 230/2016 June /7

6 Table 6: Results of the recovery of nitrogen in glycine applying mirco-kjeldahl Glycine m Sample [g] V Sample [ml] %N Gly Recovery [%] Sample Sample Sample Sample Average [%] Rsd [%] The mean blank volume (V Blank ) was ml (n = 2) Nitrogen determination in bovine serum albumin The results of the determination of nitrogen content in bovine serum albumin, according to the standard-kjeldahl method and the mirco-kjeldahl method are presented in Tables 7 and 8. Table 7: Results of the determination of nitrogen with standard-kjeldahl (declared nitrogen content 1.6 mg/ml). Sample V Sample [ml] V Titrant [ml] N [mg/ml] Result Result Result Result Average [mg/ml] 1.63 Rsd [%] 1.86 The mean blank volume (V Blank ) was ml (n = 2). Table 8: Results of the determination of nitrogen with mirco-kjeldahl (declared nitrogen content 1.6 mg/ml). Sample V Sample [ml] V Titrant [ml] N [mg/ml] Result Result Result Result Average [mg/ml] 1.59 Rsd [%] 1.57 The mean blank volume (V Blank ) was ml (n = 2). 6. Comparison standard-kjeldahl and mirco-kjeldahl This application note is based on standard-kjeldahl and mirco-kjeldahl. The experimental differences between the standard- and micro-kjeldahl are shown in Table 9. Table 9: Differences to standard-kjeldahl and mirco-kjeldahl Standard-Kjeldahl Sample tube 300 ml 100 ml Catalyst 3.5 g K 2SO 4, g CuSO 4*5H 2O, g TiO 2 Sulfuric acid 10 ml 3 ml Sodium hydroxide 60 ml (Conc. 32 %) Mirco-Kjeldahl 1.5 g K 2SO g CuSO 4*5H 2O g TiO 2 30 ml (Conc. 32 %) Digestion time 120 min 60 min Application Note 230/2016 June /7

7 7. Conclusion The determination of nitrogen in pharmaceutical products as bovine serum albumin using the SpeedDigester K-436 and KjelFlex K-360 provides reliable and well reproducible results. The found results correspond very well to the labelled value of the sample analyzed with low relative standard deviations. There are no differences between the results obtained with standard-kjeldahl and mirco- Kjeldahl. The time applying the mirco-kjeldahl method is only 60 min. In addition, the micro-kjeldahl method is a good alternative for homogeneous samples with a high nitrogen content. Micro-Kjeldahl helps to speed up the time and reduces the amount of chemicals needed for the determination, thereby saving money and resources. 8. References [1] AOAC Microchemical Determination of Nitrogen (Micro-Kjeldahl Method). [2] Standard of Chinese pharmacopeia (version 2015): Nitrogen determination in Medicine. Application Note 230/2016 June /7