Total Kjeldahl Nitrogen Determination for Quality Control - Buchi
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Total Kjeldahl Nitrogen Determination for
Quality Control
Nowadays, within the framework of Good Manufacturing Practice (GMP), Quality Control (QC) is
vital for both raw materials and products in pharmaceutical manufacturing. Multiple compounds
are processed and combined to deliver pharmaceutical products to the customer, and each of
these compounds needs to be safe and controlled. A reliable primary method is required that
covers a broad range of possible testing subjects. To achieve this objective on an economical
scale, the method of choice for pharmaceutical QC is Total Kjeldahl Nitrogen (TKN).
Background Information – Compounds in Pharmaceutical Manufacturing
The pharmaceutical manufacturing process uses numerous compounds present in different
physical forms, such as powders, granulates, and liquids. The primary compound classes are
shown in Fig. 1 and covered by TKN determination. The only prerequisite they have to fulfill is
that their chemical structure contains nitrogen atoms, which is the case for most Active
Pharmaceutical Ingredients (APIs) and other compounds. According to Njardarson et al.1 84% of
small molecule drugs approved by the FDA contain at least one nitrogen atom and are therefore
eligible for this analytical method.
Fig. 1: Fields of application for TKN determination.
Furthermore, BUCHI’s Kjeldahl instrumentation completely fulfills the described methods in the
European Pharmacopoeia and US Pharmacopeia norms.
The European Pharmacopoeia (Ph.Eur.) describes two Kjeldahl methods. One for nitrogen (2.5.9)
and another for total protein (2.5.33(7)) determination. The method 2.5.33(7) is based upon
method 2.5.9, which describes commercial instrumentation for nitrogen and total protein analysis.
The United States Pharmacopoeia (USP) describes two methods for nitrogen determination. In
USP 461 method I, the standard distinguishes between samples containing nitrates/nitrites and
samples without these components. In USP 461, method II, accelerated digestion using hydrogen
peroxide (H O ) as an oxidizing agent, is described, followed by boric acid titration.
2 2
1
Vitaku, E.; Smith, D. T.; Njardarson, J.T.; Journal of Medicinal Chemistry 2014, 57, (24), 10257–10274
Application Review 03/2020 27 January 2021 1/6
General Method Overview – TKN Determination TKN analysis determines the content of both organic and inorganic forms of nitrogen in pharmaceutical compounds. Before employing this method, it must be understood that not all chemical groups containing nitrogen are suitable for this analytical method (Fig. 2). Fig. 2: Detectable and non-detectable chemical groups in the molecular structure The analysis starts with the acid digestion of the sample, converting organic nitrogen to ammonia. The sample needs to be boiled in concentrated sulfuric acid, potassium sulfate, and a copper catalyst to convert the organic nitrogen to ammonia (Fig. 3). Fig. 3: Digestion Process via block heating The second part of the method involves steam distillation. The pH of the digestate must be raised to 9.5 by adding concentrated sodium hydroxide. At this pH, ammonia gas forms. The ammonia gas is then transferred through steam distillation into the acidic absorbing solution, namely diluted boric acid, and converted to ammonium (Fig. 4). Nitrogen concentrations within the receiving solution can then be determined using classical potentiometric or colorimetric electrode determination methods. Application Review 03/2020 27 January 2021 2/6
Fig. 4: Steam Distillation Process. ① Steam is inducted into the sample tube ② Steam passes through the sample ③ Analyte passes through the splash protector by means of steam distillation ④ Volatile components are condensed and collected in the receiver Key Parameters to Consider – Titration Potentiometric vs. Colorimetric Titration The highly regulated QC environment requires authoritative qualifications, compliant titration techniques, and traceable data management. For the detection of pH, two different techniques can be applied: potentiometric and colorimetric titration. Potentiometric titration is based on the measurement of electrical voltage using a pH electrode. For colorimetric titration, an indicator is used to measure pH-dependent color changes with a colorimetric sensor that measures the absorbance of light. When using colorimetry, it is necessary to determine the setpoint (patented) daily, prior to blank and sample measurements. The setpoint defines the endpoint of the subsequent titrations. The protection mesh must be installed to shield the measuring beam from air bubbles. Two mixed indicators are supported, the Sher indicator and bromocresol green-methyl red mixed indicator. The boric acid solution’s pH has to be adjusted, dependent on the chosen indicator and boric acid concentration. Otherwise, the setpoint might not be detected. Due to the Sher indicator’s excellent properties, the titration speed is much higher compared to bromocresol green-methyl red. Application Review 03/2020 27 January 2021 3/6
Advantages Disadvantages
Potentiometric ∙ Lower detection limit2 ∙ Daily calibration required
direct pH ∙ IntelliDist and back titration ∙ Electrode lifetime of 6 – 12 months
measurement possible ∙ Storage of pH electrode in saturated KCl
∙ No calibration of the sensor ∙ IntelliDist3 and back titration not possible
Colorimetric necessary ∙ Indicator required (Sher or bromocresol green-
detection of ∙ Longer lifetime of the sensor methyl red indicator only!)4
color change (approx. ∙ protection mesh required to protect the sensor from
4 years) bubbles and to avoid titration errors
∙ Endpoint visible ∙ Daily determination of the Setpoint necessary
Boric Acid vs. Back Titration
Two titration types are commonly used: boric acid and back titration
Boric Acid Titration
Boric acid titration is most commonly used as it involves direct detection and allows for automation
without additional equipment. The concentration of captured ammonium ions in the boric acid is
determined using an acid-base titration commonly using standard sulfuric or hydrochloric acid
solutions. Depending on the number of ammonium ions present, concentrations in the range of
0.01 N to 0.5 N are used. The receiving solution is boric acid, adjusted to a pH of 4.65 to capture
the nitrogen carried over as ammonia during steam distillation. The subsequent endpoint titration
is performed with an acid titration solution. This titration type does not require an accurate dosage
of boric acid.
Back Titration
The alternative type of titration is back titration. Here the receiving solution is a standardized acid
of which an accurate volume is dispensed into the receiving vessel. After collecting the ammonia,
the excess acid is titrated with a basic titration solution at pH 7.00. Depending on the regulations
being followed, another endpoint pH may be chosen.5 If the use of boric acid has to be avoided,
back titration is the procedure of choice.
Advantages Disadvantages
Boric acid titration ∙ Easy and approximate ∙ Requires the use of Boric acid
Receiver = H3BO3 dosage of the receiver
Titrant = H2SO4 / HCl
Endpoint = 4.65
Back titration ∙ No boric acid required ∙ Additional dosing unit required for accurate
Receiver = H2SO4 dosage of the receiving solution
Titrant = NaOH ∙ More expensive, as additional dosing unit
Endpoint = 7.00 and more volumetric solutions are required
2
Due to lower concentrated titrants that can be used - see also best@buchi 58/2010 “Low detection and quantification
limits
3
IntelliDist eliminates errors caused by a cooled down instrument. The countdown of the set distillation time only starts
after the operating temperature is attained
4
Optimal point of inflection at 610 nm
5
European Pharmacopoeia (Ph. Eur.) 2.5.9
Application Review 03/2020 27 January 2021 4/6Additional Tips & Tricks – Selection of Accessories IR-digestion vs. Block-digestion The direct physical contact of the block’s metal surface with the glass causes a flux of heat energy, higher in block digestion than the heat flow in an IR digester that relies on convection and irradiation. The advantage of block digestion is the larger number of samples that can be digested at one time, enhancing productivity. With a block digester, 20 samples can be placed into one rack. The largest number of samples that can be digested with an IR digester is 12. Kjeldahl Tablets Today’s requirements for a catalyst mixture lie in the shortening of digestion time and the possibility of dealing with foamy samples and protecting the user and the environment. Therefore, we have multiple options available to fit individual needs. Boric Acid Two concentrations of boric acid are recommended for Kjeldahl: ∙ 2 % (w/v) boric acid for nitrogen contents ≤ 6.75 mg N absolute ∙ 4 % (w/v) boric acid or nitrogen contents ≥ 6.75 mg N absolute Indicators for Boric Acid Titration Detecting the endpoint during colorimetric titration requires adding an indicator to the boric acid. Two different indicators can be used for colorimetric titration: 1. Sher mixed indicator 2. Bromocresol green-methyl red mixed indicator Sample Changer The ultimate advantage of block digesters is the compatability of the rack, which can be used in sample changers (KjelSampler). Block digesters assist in the automation of the subsequent distillation and titration processes by making use of the KjelSampler sample changer. PC Software The KjelLink PC software is a monitoring, preparation, and evaluation software to be used when working with the distillation unit. ∙ Distillation and titration processes in progress can be observed online ∙ Preparation of analyses and export to the distillation unit for further processing ∙ Recorded Kjeldahl results can be imported and saved for further use ∙ Recalculation of results Compliance to 21 CFR Part 11 – The Pharma Package Our firmware is designed to achieve compliance with 21 CFR Part 11. ∙ User administration is supported by three user levels (Administrator, Lab-Manager, and Operator) with predefined rights ∙ All electronic records, or their copies, are human-readable, and a backup procedure is offered for data recovery ∙ The instrument permanently tracks all quality-relevant events and automatically stores it in the audit trail. Entries are protected from changes by any user ∙ Electronic signatures are unambiguously linked to electronic records and prevented from falsification. IQ/OQ available Installational and Operational Qualification (IQ/OQ) are available for the complete KjelMaster portfolio. Only certified employees perform and document the IQ/OQ qualification. Furthermore, BUCHI arranges individual training courses through all application fields. Application Review 03/2020 27 January 2021 5/6
Application Examples The application protocols for pharmaceutical compounds are as diverse as the fields of the application themselves. Below are some examples of how to determine the nitrogen content of pharmaceutical products. https://static1.buchi.com/sites/default/files/2016_264_AN_Pharmaceutical_active_peptide s_0.pdf https://static1.buchi.com/sites/default/files/2016_279_AN_Nitrogen_deter mination_in_toxo ids_0.pdf https://static1.buchi.com/sites/default/files/230_2016_Application_Note_Nitrogen_determi nation_in_bovine_serum_albumin.pdf https://static1.buchi.com/sites/default/files/AN_045_2010_Nitrogen_Determination_Pharm aceuticals_Semi-Micro.pdf Application Review 03/2020 27 January 2021 6/6
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