Applications

Aseptic identification of Polysorbate 20 and 80 through amber bottles using 1064 nm Raman, reducing fluorescence and enabling reliable raw material verification.

The analytical challenge treated by the present work consists in detecting sub-ppb concentrations of low-molecular-weight amines in the presence of strongly retained cationic drugs by using ion chromatography (IC) with upstream inline coupled-column matrix elimination (CCME). In contrast to direct-injection IC, where the late elution of strongly retained drugs requires eluents with added acetonitrile, the CCME technique uses two preconcentration columns in series. In an «inverse matrix elimination step, cationic drug and target amines are trapped on a high-capacity and a very-high-capacity preconcentration column, respectively. During amine determination, a rinsing solution flushes the drug to waste. This significantly shortens the analysis time and improves sensitivity as well as selectivity. Besides the determination of monomethylamine in Nebivolol hydrochloride discussed here, the CCME technique is a promising tool for detecting further low-molecular-weight amines in a wide range of drugs.

The poster describes the water determination in pharmaceuticals using the Karl Fischer oven technique.

Pharmaceutical analysis guarantees drug safety by providing information on the identity, content, quality, purity, and stability of pharmaceutical products using analytical chemistry. Ion chromatography (IC) offers a broad range of pharmacopeia-compliant applications for quality control, monitoring, and improving drug manufacturing.As a very accurate and versatile technique, IC meets the requirements of many pharmaceutical applications. IC is a USP-accepted standard method for the determination of active pharmaceutical ingredients (APIs), excipients, impurities,pharmaceutical solutions as well as pharmaceutical starting materials, finished pharmaceutical products (FPPs) and even body fluids.This poster describes some typical examples.

This poster presented jointly with USP at AAPS meeting shows, that we successfully developed and validated a single IC procedure for potassium assay and identification in potassium bicarbonate and potassium chloride for effervescent oral suspension. The optimized chromatographic conditions could be used for other cationic impurities, such as magnesium, calcium, sodium, and ammonium in potassium bicarbonate and potassium chloride for effervescent oral suspension. Single chromatographic method for assay and identification simplifies the overall QA/QC workflow.

This poster presented jointly with USP at AAPS meeting shows, that we successfully validated an IC method to determine chloride and sulfate in drug substances, potassium bicarbonate and potassium carbonate. The proposed IC method overcomes limitations of the turbidimetry/visual comparison methods.

A high throughput automated system was developed to determine pH of culture media using a pH module equipped with an external flow cell. A custom septum-piercing, vented needle was developed to accommodate the shape and size of the customer sample vials. For this application, both accurate and precise pH measurements were required. The data presented in this document was collected by a customer as a part of their validation process and was provided for use with their consent.

This document explains how to measure Na ion concentration in diverse matrices with a sodium ion-selective electrode (Na-ISE) using direct measurement and standard addition.

This Application Bulletin describes the determination of mercury by anodic stripping voltammetry (ASV) at the rotating gold electrode. With a deposition time of 90 s, the calibration curve is linear from 0.4 to 15 μg/L; the limit of quantification is 0.4 μg/L.The method has primarily been drawn up for investigating water samples. After appropriate digestion, the determination of mercury is possible even in samples with a high load of organic substances (wastewater, food and semi-luxuries, biological fluids, pharmaceuticals).

Potentiometric titration is an accurate method for determining chloride content. For detailed instructions and troubleshooting tips, download our Application Bulletin.

This Application Bulletin describes a voltammetric method for the determination of aluminum in water samples, dialysis solutions, sodium chloride solutions and digestion solutions (e.g. of lyophilisates). The method utilizes the complexation of the Al3+ ion by Calcon (Eriochrome blue black R). The formed complex can easily be reduced electrochemically at 60 °C. The limit of quantitation depends on the purity of the reagents used and is approx. 5 µg/L.

This Bulletin describes three potentiometric, one photometric, one thermometric and one conductometric titration method for sulfate determination. The question of which indication method is the most suitable depends primarily on the sample matrix.Method 1: Precipitation as barium sulfate and back titration of the Ba2+ surplus with EGTA. Use of the ion-selective calcium electrode as indicator electrode.Method 2: As with Method 1, although with the electrode combination tungsten/platinum.Method 3: Precipitation titration in semi-aqueous solution with lead nitrate in accordance with the European Pharmacopoeia using the ion-selective lead electrode as indicator electrode.Method 4: Photometric titration with lead nitrate, dithizone indicator and the Optrode 610 nm, particularly suitable for low concentrations (up to 5 mg SO42- in the sample solution).Method 5: Thermometric precipitation titration with Ba2+ in aqueous solution, particularly suitable for fertilizers.Method 6: Conductometric titration with barium acetate in accordance with DIN 53127

Sulfide and sulfite can be determined polarographically without any problems. For sulfide, polarography is performed in an alkaline solution, for sulfite in a slightly acidic primary solution. The method is suitable for the analysis of pharmaceuticals (infusion solutions), wastewater/flue gas water, photographic solutions, etc.

Cinchocaine (dibucaine) is used in the form of ointments or injection solutions as a local anaesthetic. Its base is soluble in diethyl ether; its hydrochloride, on the other hand, is insoluble in diethyl ether but easily soluble in water. This Bulletin describes the determination of cinchocaine in ointments, creams and injection solutions by means of differential pulse polarography. An acetate buffer pH = 4.8 is used as the supporting electrolyte. The limit of quantitation and the linear working range of the method are given. The necessary sample preparation steps are also dealt with in this Bulletin.

The present Bulletin offers an overview of the multitude of surfactants and pharmaceuticals that can be determined with potentiometric titration. Metrohm provides five different surfactant electrodes for indicating the titration endpoint: the Ionic Surfactant, the High Sense, the Surfactrode Resistant, the Surfactrode Refill and the NIO Surfactant electrode. The manufacture of the respective titrants and their titer determination are described in detail. In addition to this, the Bulletin contains a tabular overview of more than 170 proven applications from the area of surfactant and pharmaceutical analysis. This guideline leads you reliably to your destination: At a glance you can see from the table which surfactant electrode and which titrant are optimally suitable for your product.

On the basis of a multitude of practical examples, this Bulletin describes the potentiometric two-phase titration of ionic surfactants in raw materials and many other formulations.Two surfactant electrodes – the Surfactrode Resistant and the Surfactrode Refill – make it possible to perform this type of surfactant titration, analogous to the classic "Epton titration", with a high degree of automation. The achieved results correlate very well with those of Epton titration. The toxic, carcinogenic and environmentally hazardous chloroform can be replaced by other solvents such as methyl iosbutyl ketone or n-hexane.

The present Application Bulletin contains NIR applications and feasibility studies using NIRSystems devices in the pharmaceutical industry. Qualitative and quantitative analyses of a wide variety of samples are part of this bulletin. Each application describes the instrument that was originally used for the analysis, as well as the system recommended for the analysis and the results that were achieved thereby.

This Bulletin describes the automatic measurement of conductivity in water samples with low electrical conductivity in accordance with USP<645>. Conductivity measurement is demonstrated on the example of ultrapure water, which is used, among other things, to produce injection solutions in the pharmaceutical sector.

Determination of sodium, potassium, calcium, and magnesium in a drip feeding formula using cation chromatography with direct conductivity detection and dialysis as sample preparation.

Determination of betaine in the presence of standard cations in an Echinacea product using cation chromatography with direct conductivity detection.

Determination of zinc, sodium, potassium, magnesium, and calcium in an infusion solution using cation chromatography with direct conductivity detection.

A cardioplegic solution protects the ischemic myocardium from cell death. It is applied together with hypothermia e.g. in open heart surgery. Here the simultaneous determination of aspartic acid, glutamic acid, tris(aminomethyl)aminomethane (TRIS), sodium and potassium in such a solution is given. The two amino acids can be determined as they are partially in the triple protonated ammonium form at the eluent pH. Determination is achieved by direct conductivity detection.

Tris(hydroxymethyl)aminomethane (TRIS) is often used in life science applications and its purity must be monitored. This analysis is possible with ion chromatography.

Potassium citrate and citric acid oral solutions act as systemic alkalizers. Potassium assays, validated per USP <621> and <1225>, use IC with L76 cation-exchange columns.

The iodine content in numerous iodized X-ray contrast media (ICM) is around 50% and is determined with great accuracy using Combustion Ion Chromatography. Large quantities of H2O2 (1,000 mg/L) are required for complete absorption of the iodine. Similarly, the concentration of the internal standard should be 50 mg/L. The water content of the X-ray contrast media is determined by means of Metrohm's Karl Fischer oven method and incorporated in the final calculation.Keyword: pyrohydrolysis

Compounded injections of sodium bicarbonate are sterile solutions for correcting metabolic acidosis and other conditions requiring systemic alkalinization. Compounded injections of sodium phosphates serve as a phosphate source to either prevent or correct hypophosphatemia in patients with restricted oral intake. Ion chromatography (IC) with suppressed conductivity detection is the standardized way to accurately quantify sodium in these solutions.

Ion chromatography (IC) provides an automated, fast, and sensitive solution to accurately quantify cationic and anionic components including acetate simultaneously. This comprehensive approach makes IC an economic alternative to traditional techniques for the quality control of pharmaceutical solutions like haemodialysis concentrates. Ease-of use, accuracy, and the high-throughput of IC increase productivity and comply with the demands of modern routine and research labs.

Iron sucrose injections are used during the treatment of iron deficiency anemia. They contain a mixture of ferric iron (Fe3+) and ferrous iron (Fe2+). Ferrous iron content may be determined by subtracting the ferric iron content from the total determined iron content. Yet, this increases the measurement error due to error propagation. Alternative determination of iron(II) with cerium(IV) by potentiometric titration may be hampered, as the equivalence point cannot be determined unequivocally. Determination by thermometric titration is a more robust and therefore more reliable alternative, as this method is unaffected by the sample matrix. Here, the endpoint of the titration is indicated by a fast responding thermometric sensor. Endpoint detection is further improved by spiking the sample with 0.2% ammonium iron(II) sulfate (FAS), increasing the reliability of the determination. Compared to potentiometric titration, thermometric titration is faster and more convenient as no sensor maintenance is required. One determination takes about 2–3 minutes.

The water content of lyophilisates contained in vials is determined by Karl Fischer titration. Conditioned solvent (methanol) is injected into the vial to dissolve the sample and extract the water (ultrasonic bath). Afterwards the contents of the vial are transferred to the titration vessel to carry out the automatic determination.

This application note describes the water content determination in penicillin by using volumetric Karl Fischer titration. Unwanted side reactions can be avoided by using special solvent mixtures.

Determination of acetate, lactate, and chloride in electrolyte solutions using anion chromatography with direct conductivity detection.

Determination of acetate, chloride, citrate, and sulfate in a concentrate of an infusion solution using anion chromatography with direct conductivity detection. Non-suppressed IC is used to avoid interferences by the amino acids.

Determination of acetate, chloride, and malate in an infusion solution using anion chromatography with direct conductivity detection.

Determination of acetate, phosphate, chloride, and citrate in an infusion solution using anion chromatography with direct conductivity detection.

Heparin acts as an efficient anticoagulant and, in addition to direct injection, is also used as a lock-flush solution for rinsing catheters. Vis-NIR spectroscopy can be used to determine the strength of contaminated and purified heparin. This Application Note demonstrates that heparin strength can be determined reliably with Vis-NIR spectroscopy.

Polyvinyl alcohol (PVA) is a linear polymer, used in a variety of medical products (e.g. eye drops). Here, the degree of alcoholysis is an important index for the water solubility, viscosity, and adhesion of the product. The degree of alcoholysis is defined as the percentage of hydroxyl functional groups compared to the total functional groups accessible in the molecule. Conventional alcoholysis determination can take up to six hours per sample. Compared to the primary method, analysis with near-infrared spectroscopy (NIRS) only takes one minute. The following application note describes the determination of the degree of alcoholysis by NIRS.

Cell fermentation processes are a reliable production method for small molecules and protein-based active pharmaceutical ingredients (APIs). The fermentation process requires monitoring of many different parameters to ensure optimal production. These quality parameters include pH, bacterial content, potency, glucose, and concentration of reducing sugars. Traditional laboratory analysis takes a significant amount of time and requires different analytical techniques to monitor these different quality parameters. Near-infrared spectroscopy (NIRS) offers a faster and more cost-efficient alternative to traditional methods for the determination of critical parameters in fermentation broths at any stage of the fermentation process.

This Application Note shows how easy it is to determine water content in the pharmaceutical excipient lactose with reagent-free near-infrared spectroscopy.

Traditionally, catecholamines are separated using reversed-phase chromatography followed by amperometric detection. This Application Note describes the determination of catecholamines in an emergency medication for life-threatening allergic reactions.

This Process Application Note presents a method to accurately monitor lactic acid and glucose inside a bioreactor in «real-time» with the 2060 Raman Analyzer from Metrohm Process Analytics.

This Application Note shows the rapid, non-destructive identification of isopropyl alcohol from two manufacturers using Raman spectroscopy following the creation of a suitable library. The measurements with the hand-held Raman spectrometer Mira M-1 require no sample preparation and provide immediate results that identify the samples unambiguously.

Low frequency Raman spectroscopy extends conventional Raman analysis by capturing vibrational modes down to 65 cm-1, enabling deeper insights into molecular structure, protein characterization, polymorph identification, and phase changes.

Determination of bromide and sulfate using anion chromatography with conductivity detection after chemical suppression.

Determination of acetate, chloride, phosphate, and succinate in an infusion solution using anion chromatography with conductivity detection after chemical suppression.

Determination of chloride, nitrite, bromide, nitrate, and sulfate in water for infusion solution production using anion chromatography with conductivity detection after chemical suppression.

Determination of acetate, chloride, sulfate, and citrate in a pharmaceutical product using anion chromatography with conductivity detection after chemical suppression and dialysis for sample preparation.

Nadroparin is a low-molecular-weight heparin used as a anticoagulant to prevent thrombosis. The determination of sulfate in the sulfur-containing anticoagulant is performed to control the degradation of the product. Thanks to the absence of interfering peaks close to sulfate, a short column could be used.

β-glycerophosphate and malate are determined in a pharmaceutical formulation. Excellent separation of β-glycerophosphate and malate from α-glycerophosphate and phosphate is possible with the aid of a carbonate eluent and the Metrosep A Supp 7 - 250/4.0 column.

Fluoride is commonly used in dental products to help prevent tooth decay. If the products are intended to prevent the formation of cavities (carries), then it is regulated by the US Food and Drug Administration (USFDA) as an Over-the-Counter (OTC) Drug. Previously, the assay of Fluoride in oral solution was done by Ion selective electrode and identification was done by tedious wet chemistry method. USP has updated this monograph for Assay and identification tests with Ion Chromatography using L46 packing. The Metrosep A Supp 1 - 250/4.6 column fulfills all USP acceptance criteria. It therefore is a viable alternative separation column for the determination of sodium fluoride in oral solutions.

Dental care products often contain sodium fluoride as an active ingredient. Manufacturers use the United States Pharmacopeia and National Formulary (USP-NF) Monograph «Sodium Fluoride» to quantify sodium fluoride and its anionic contaminants chloride and acetate in these products. The validated USP method proposes ion chromatography (IC) with suppressed conductivity detection to carry out the fluoride assay as well as the impurity determination in a single chromatogram.

Sodium fluoride gel for pharmaceutical use needs to comply with USP requirements. The actual monograph (USP 42) uses two different methods for the identification and the assay. Ion chromatography allows the analysis of these two parameters in one single determination. In the course of the USP monograph modernization, this ion chromatographic approach makes this type of analysis even easier.

Stannous fluoride gel for pharmaceutical use needs to comply with USP requirements. The actual monograph (USP 42) utilizes two different methods for the identification and the assay. Ion chromatography allows the analysis of these two parameters within a single determination. In the course of the USP monograph modernization, this ion chromatographic approach makes this type of analysis even easier.

Sodium fluoride and phosphoric acid gel for pharmaceutical use need to comply with USP requirements. The actual monograph (USP 42) uses two different methods for the identification and the assay. Ion chromatography allows the measurement of these two parameters within a single determination. In the course of the USP monograph modernization, this ion chromatographic approach makes this type of analysis even easier.

In the course of USP column equivalency tests, the Metrosep A Supp 3 - 250/4.0 is applied for the assay of citric acid/citrate and phosphate according to USP general Chapter <345>. This report shows that the Metrosep A Supp 3 - 250/4.0 column is equivalent to packing L61 required in USP general Chapter <345>.

An ion chromatography (IC) assay with suppressed conductivity detection is the standardized way to accurately quantify phosphate in phosphates compounded injections.

Anticavity pharmaceuticals like sodium fluoride and acidulated phosphate topical solution products require strict quality control. This Application Note outlines the fluoride IC assay as described in the USP Monograph Sodium Fluoride and Acidulated Phosphate Topical Solution.

In severe cases of cyanide poisoning, sodium nitrite is used along with sodium thiosulfate for treatment. This Application Note describes the nitrite ion chromatography assay with the Metrosep A Supp 4 column and suppressed conductivity detection. This column equivalency study was in cooperation with the USP according to the USP General Chapter <621>.

Determination of tranexamic acid in injection solutions by nonaqueous potentiometric titration with perchloric acid using a glass electrode.

Determination of acetate, chloride, and phosphate in an infusion solution by potentiometric titration with sodium hydroxide after conversion of the anions to the corresponding acids.

The purity of sulfanilamide was determined by means of automatic potentiometric titration using sodium nitrite as the titrant. The solution was spiked with potassium bromide, because bromide ions catalyze diazotization titration.

This Application Note details the determination of acid-neutralizing capacity (ANC) in several pharmaceutical samples in compliance with USP<301> standards.

Sulfonamides are drugs used to treat allergies and cough. They also have some antifungal and antimalarial activities. USP<451> describes nitrite titration method for the determination of numerous pharmacopeial sulfonamide drugs and their dosage forms as well as of other pharmacopeial drugs with, for example, hydrazide (e.g., in isoniazid) and amine ester groups (e.g., in procaine) or amide derivatives of amino acids.Here, for illustrating the analysis of the latter, the assay of the diagnostic agent aminohippuric acid is described.

Kjeldahl method is used to determine the nitrogen content in organic and inorganic samples. Kjeldahl consists of three steps: digestion, distillation, and titration. During the catalytic digestion step, organic nitrogen is converted into ammonium. Sodium hydroxide is added just before the distillation step for converting ammonium into ammonia. Through steam distillation the latter is transferred into the receiver vessel containing an absorbing agent (e.g., boric acid). Finally, the separated ammonia is titrated against sulfuric acid. Protein content in samples can also be determined from the nitrogen content obtained by Kjeldahl setup. USP describes the titration method to determine nitrogen content in organic products using Kjeldahl nitrogen setup. This Application Note illustrates nitrogen determination in heparin sodium.

Vitamin C, also known as ascorbic acid or L-ascorbic acid, is an essential nutrient involved in the repair of tissues and the enzymatic production of certain neurotransmitters. It is required for the functioning of several enzymes and immune performance, and is also an important antioxidant. This nutrient is found in many foods and is often used as a dietary supplement.USP general chapter <580> describes a titration technique to determine the assay of Vitamin C as ascorbic acid, sodium ascorbate, and calcium ascorbate dehydrate, or their mixture in finished dosage forms as capsules, tablets, and oral suspensions. This Application Note demonstrates the Vitamin C determination in water-soluble vitamin tablets. The methodology can also be applied for oil-soluble vitamin or mineral tablets, as well as oil- and water-soluble vitamin or mineral capsules.

The determination of amino acid is an important task in pharmaceutical and biochemical applications. A binary gradient separates in this example 17 amino acids of a commercially available standard solution. The post-column reaction with ninhydrin requires a temperature of 120 °C, while the samples need to be cooled for stability.

Aluminum (as gel or salt) is used in vaccines as an adjuvant. This helps to get a stronger immune response. The amount of aluminum in vaccines is regulated e.g., by USP. According to USP maximum amounts of Al3+ in a vaccine dose lay between 0.85 and 1.25 mg. This work describes the determination of aluminum as the 8-hydroxyquinoline complex by ion chromatography with UV/VIS detection.

Manufacturers and laboratories must use validated methods to determine the zinc content in skin care products and pharmaceutical supplements to meet strict quality standards set by the United States Pharmacopeia and National Formulary (USP-NF). USP-NF has updated the zinc monograph (General Chapter <591>, Zinc Determination) and replaced the existing identification procedure of titration with ion chromatography (IC). The analysis involves separating Zn using a Metrosep A Supp 10 column followed by post-column reaction using 4-(2-pyridylazo)resorcinol and detection at 530 nm.

Determination of clothiapine in a pharmaceutical standard.

Determination of artemisinin and artesunate in a standard.

Determination of ß-propiolactone in vaccine.

Thiomersal (also called thimerosal) is a mercury containing organic molecule that has been widely used as preservative for vaccines and eye drops. It is very effective, even in very low concentrations, against a wide range of microorganisms and viruses. To reduce the risk for consumers the maximum concentration of mercury in the products is limited by the authorities. Polarography or voltammetry can be used to accurately determine the concentration of thiomersal in vaccines or other cosmetic and pharmaceutical solutions (such as eye drops). The method is simple to perform, specific, and free of interferences.

Determination of cysteine and cystine in an infusion solution.

Iron sucrose injection is a dark brown liquid which contains sucrose and iron(III) hydroxide in an aqueous solution, commonly used for the treatment of iron deficiency anemia. As a medical product, iron sucrose is subject to strict controls. Among other tests, the U.S. Pharmacopeia (USP) requires to monitor the limit of Fe(II) in the iron sucrose injection solution by polarography. The benefit of polarography is that Fe(II) and Fe(III) show signals at different potentials, and therefore an easier determination of Fe(II) without a previous separation of the two oxidation states is possible. The 884 Professional VA together with the viva software allows a straightforward determination of the Fe(II) content of iron sucrose injection solution following the requirements of the USP. The Fe(II) content is automatically calculated and stored in a database together with all relevant determination and calculation parameters.

Sodium pertechnetate (99mTc) radiopharmaceuticals are widely used in medical imaging diagnostic procedures to help diagnose a large number of diseases affecting the bones and major organs. These radiopharmaceuticals are usually prepared from cold kits consisting of several ingredients, including a reducing agent. Sn(II) is a typical reducing agent which reduces the Tc(VII) that is added to the cold kit to a lower oxidation state which then forms the stable organic complex.For quality control, the tin content has to be determined in the kit vial. Sn(II) can be selectively determined using differential pulse polarography. Polarography is a straightforward, sensitive, selective, and interference-free method for the determination of mg/L levels of Sn(II) in radiopharmaceuticals.

This article describes the investigation using ion chromatography and subsequent inductively coupled plasma mass spectronomy (ICP/MS) to determine the extent to which the iron(III) flocculation carried out in the context of wastewater treatment releases toxic gadolinium(III) ions as the result of recomplexing.

The coupling of ion chromatography and inductively coupled plasma mass spectrometry (ICP/MS) leads to a high-performance measurement system that masters several particularly challenging analyses. It enables for example reliable determination of element compositions, oxidation states and chemical bonds. This information is used, for example, for assessing the toxicity of medications, environmental and water samples as well as foods and beverages.

The pharmaceutical industry is very likely more comprehensively regulated that any other branch of industry. It therefore requires analytic methods that meet the requirements of regulations while at the same time being practical. This applies in particular for large sample quantities, such as are encountered with incoming goods inspections, for example. It is here that particularly rapid and simple analysis methods are called for which make routine analyses simpler and more efficient. This White Paper describes some of the most important regulations in the pharmaceutical analysis and shows how Vis-NIR spectroscopy can solve analytic problems in the pharmaceutical industry in accordance with regulations.

Chemometrics is a powerful tool widely used for method development in the pharmaceutical industry. This whitepaper describes the lifecycle of multivariate models and summarizes the workflow of the development of chemometrical models according to the new USP chapter <1039>.

Ion measurement can be conducted in several different ways, e.g., ion chromatography (IC), inductively coupled plasma optical emission spectrometry (ICP-OES), or atom absorption spectroscopy (AAS). Each of these are well-established, widely used methods in analytical laboratories. However, the initial costs are relatively high. In contrast, ion measurement by the use of an ion-selective electrode (ISE) is a promising alternative to these costly techniques. This White Paper explains the challenges which may be encountered when applying standard addition or direct measurement, and how to overcome them in order for analysts to gain more confidence with this type of analysis.

This white paper summarizes the steps involved in converting an existing manual titration procedure to semi-automated or automated titration procedures. It discusses topics such as selecting the right electrode and titration mode. For a better understanding, the discussion topics are illustrated with three examples.

The objective of validation of an analytical procedure is to demonstrate that it is suitable for its intended purpose. Recommendations for the validation of analytical methods can be found in ICH Guidance Q2(R1) Validation of Analytical Procedures: Text and Methodology and in USP General Chapter <1225> Validation of Compendial Procedures. The goal of this white paper is to provide some recommendations for the validation of titration methods.

Haemophilus influenzae type B (Hib) is a major cause of bacterial meningitis in children in many countries. The capsular polysaccharide (PS) of Hib plays an important role in the virulence of the organism. The polysaccharide capsule hides cell surface components from elements of the mammalian immune system, such as antibodies and complement proteins that otherwise would activate mechanisms to kill the pathogen. Vaccines require rigorous characterization and assays to ensure final product quality and consistency. For glycoconjugate vaccines, it is important to measure both free and total PS to ensure the quality. A large amount of unconjugated PS may suppress immunity to the antigen. Additionally, the presence of free PS is a key indicator of process consistency. Current methods to determine PS content in vaccines such as Hib are imprecise and unreliable, especially if the vaccine contains a sugar stabilizer (e.g. lactose). Ion chromatography with pulsed amperometric detection (IC-PAD, or HPAEC-PAD) offers a simpler procedure and better sensitivity than other assays to quantify PS (here, polyribosylribitol phosphate [PRP]) in Hib vaccine.