Optimization of modified moisture measurement with Karl ...1384216/FULLTEXT01.pdfThe method for measuring the water content was the Karl Fischer titration method and the instrument

IN DEGREE PROJECT ENGINEERING CHEMISTRY,SECOND CYCLE, 30 CREDITS

, STOCKHOLM SWEDEN 2019

Optimization of modified moisture measurement with Karl Fischer to measure moisture content in freeze-dried enzyme beads for improvement of diagnostic kits

CATHARINA JAKOBSSON

KTH ROYAL INSTITUTE OF TECHNOLOGYSCHOOL OF ENGINEERING SCIENCES IN CHEMISTRY, BIOTECHNOLOGY AND HEALTH

Optimization of modified moisture measurement with Karl

Fischer to measure moisture content in freeze-dried enzyme

beads for improvement of diagnostic kits

Catharina Jakobsson

[email protected]

Master of Science in Molecular Science and Engineering

Supervisors at Cepheid: David Herthnek and Andrey Yakovlev

Examiner: Magnus Johnson

June 10, 2019

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Abstract

Cepheid provides instruments and tests for molecular diagnostic testing of bacterial andviral diseases. Their goal is to deliver a better way to improve patient outcomes by en-abling access to molecular diagnostic testing everywhere. These test kits are used in warmand humid countries which may challenge the stability of the kits. The test kits containlyophilized beads, which contain enzymes for the polymerase chain reaction, which usuallyages first. The ageing of the beads is e↵ected by the initial water content in them since itdefines the quality. Therefore, it is of great importance to have a very precise and exactwater measurement method. The degree project covers the optimization of the StandardOperating Procedure used today at Cepheid with the research question: Can optimization ofthe Karl Fischer method improve the precision when measuring the initial moisture contentof freeze-dried enzyme beads?

The method for measuring the water content was the Karl Fischer titration method andthe instrument used was the Mettler Toledo C30S Compact Karl Fischer-coulometer. Thedi↵erent optimized parameters were the sample preparation, extraction process and how themeasurement was proceeded.

It can be concluded that optimized method provides significantly di↵erent results com-pared with the old method and therefore the new method is better.

Sammanfattning

Cepheid tillverkar instrument och test for molekylar diagnostisk testning av bakteriellaoch virala sjukdomar. Malet ar att leverera en losning som leder till en forbattring for pa-tienterna genom att mojliggora tillgang till molekylar diagnostisk var an den behover utforasi valden, under stora variationer i matmiljon. Dessa testkit anvands i varma och fuktigalander vilket kan utmana stabiliteten hos produkten. Testen innehaller lyofiliserade kuloroch det ar dessa som vanligtvis aldras forst. Kulornas hallbarhet paverkas av den ursprung-liga vattenhalten, vilket definierar kvaliten. Darfor ar det mycket viktigt att ha en mycketexakt metod att mata fuktinnehallet i kulorna. Examensarbetet omfattar optimering av denstandardoperativa proceduren som anvands idag pa Cepheid och fragestallningen ar: Kanoptimering av Karl Fischer-metoden forbattra precisionen vid matning av det ursprungligafuktinnehallet i frystorkade enzym kulor?

Den metod som anvands vid matningen av vattenhalten ar Karl Fischer-titreringsmetodenoch instrumentet som anvandes var Mettler Toledo C30S Compact Karl Fischer-coulometer.De olika optimerade parametrarna var: forberedelsen av proven, extraktionsprocessen ochsjalva utforandet av matningen.

Den slutsats som kan dras efter optimeringen ar att den nya metoden ger ett mer exaktoch battre resultat an den gamla.

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Acknowledgements

I wish to thank, first and foremost, my supervisors Dr. David Herthnek and Andrey Yakovlevat Cepheid for the continuous support of my study and research, for their patience, motivation,enthusiasm, and immense knowledge.

A very special vote of thanks to Cepheid for giving me the opportunity and taking me in asa thesis student.

I also wish to thank my colleagues who acted as my operators, who took time and helpedme provide my last concluding results, QC analyst Daniel Sortebech, Research Scientist Tian Liand Research Scientist Vicente Martınez Redondo.

My sincere thanks to Sven Eriksson for his valuable guidance and sharing his knowledge.

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Contents

1 Introduction and background 6

1.1 Aim . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

2 Theory 7

2.1 Coulometric Karl Fischer (KF) analysis . . . . . . . . . . . . . . . . . . . . . . . 72.2 Generator electrode without diaphragm . . . . . . . . . . . . . . . . . . . . . . . 92.3 Water determination with KF coulometric titration . . . . . . . . . . . . . . . . . 102.4 Indication - Bipotentiometric indication . . . . . . . . . . . . . . . . . . . . . . . 112.5 KF titration for samples with very low water content . . . . . . . . . . . . . . . 112.6 Standard Operating Procedure (SOP) used today . . . . . . . . . . . . . . . . . . 12

3 Other methods for measuring the moisture content in lyophilized substances 13

3.1 Loss on Drying (LOD) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133.2 Fourier transform near-infrared (FT-NIR) spectroscopy . . . . . . . . . . . . . . 13

4 Methodology 13

5 Important factors for optimization of the KF method 13

5.1 How to work with the sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145.2 Previous research on optimization . . . . . . . . . . . . . . . . . . . . . . . . . . 14

6 Experimental 14

6.1 Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 146.1.1 Mapping of the Grant Scientific Digital Ultrasonic Bath XUB5 . . . . . . 146.1.2 Determination of extraction method . . . . . . . . . . . . . . . . . . . . . 186.1.3 Determination of extraction time on the shaking incubator . . . . . . . . 196.1.4 Finding the optimal KF reagent . . . . . . . . . . . . . . . . . . . . . . . 196.1.5 Determining the sample/solvent amount . . . . . . . . . . . . . . . . . . . 196.1.6 Comparison between the old SOP and the optimized KF method with the

help of water standards . . . . . . . . . . . . . . . . . . . . . . . . . . . . 196.1.7 Design of experiments - DOE . . . . . . . . . . . . . . . . . . . . . . . . . 20

7 Results 21

7.1 Mapping of the ultrasonic bath . . . . . . . . . . . . . . . . . . . . . . . . . . . . 217.2 Determination of extraction method . . . . . . . . . . . . . . . . . . . . . . . . . 227.3 Determination of extraction time on the shaking incubator . . . . . . . . . . . . . 227.4 Finding the optimal KF anolyte . . . . . . . . . . . . . . . . . . . . . . . . . . . . 237.5 Determining the sample/solvent ratio . . . . . . . . . . . . . . . . . . . . . . . . . 247.6 Comparison between the old SOP and the optimized KF method with the help of

water standards . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 247.7 Design of experiments - DOE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

8 Discussion 27

9 Conclusion 29

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10 APPENDICES 30

10.1 APPENDIX 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3010.2 APPENDIX 2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3710.3 APPENDIX 3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3910.4 APPENDIX 4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4210.5 APPENDIX 5 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43

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1 Introduction and background

Cepheid manufactures molecular diagnostic equipment and qualitative and quantitative testkits for a range of infectious diseases and cancer. The kits o↵er full integration of samplepreparation and PCR (Polymerase Chain Reaction) where all chemical steps are performed ina single cartridge. Each new product to be launched is tested as part of the validation of theproduct for its durability at di↵erent temperatures. The standard is that the durability is atleast one year in a storage at room temperature (which is considered 28�C). It is importantthat the kits can be manufactured with high reproducibility and quality, so durability alwaysmeets the promised requirements. Cepheid is constantly working to improve the quality of theirproducts, and it is desirable to have a precise even quality between manufactured batches, as wellas to extend the durability, and possibly even allow good shelf life at higher temperatures. Manyproducts are sold in warm and humid countries where the recommended storage environment isnot always achieved.

The kits contain, among other things, lyophilized beads with reagents. It has been shownthat it is primarily the beads which contain the enzyme for PCR, which usually ages first.Furthermore, aging is due to moisture impact and partly by slow moisture migration to thesebeads, but also because of the low amount of moisture present in the beads initially after freeze-drying.

After the manufacture and freeze-drying of the enzyme beads, their water content is tested bya method based on the Karl Fischer titration method. The method is selective for water contentand is based on oxidation of sulfur with iodine. Using the method, the enzyme beads can beevaluated and, in the rare cases where moisture content exceeds a certain limit value, they canbe discarded instead of being used and thus impairing product stability.

Preliminary studies on the amount of water in the beads and its e↵ect on the durability ofthe test kit have, among other things, shown that the Karl Fischer method could be improved toprovide a more reliable moisture measurement with less variation. The modified method uses adrier blank liquid, which will therefore constitute a minor part of the total moisture amount whenmeasured. However, the optimization of the method is still not complete, which is an importantpart of what the degree project covers. The optimization section will consist of determination ofparameters that a↵ect measurement results followed by optimization of these key factors usingDOE (Design of Experiments).

The study evaluated di↵erent beads, which are dependent on a relatively sensitive enzymewhich is widely used in warmer countries. The experience from the study will then be used fordi↵erent products and will be valuable in product development.

1.1 Aim

After optimization of the modified moisture measurement method had been completed, variousenzyme beads of varying moisture content were be produced. The water content of these beadswill be measured accurately. Furthermore, the optimized method was verified on several di↵erenttypes of beads, compared to the current method and provide a basis for producing a SOP(Standard Operating Procedure) for the new method. With an improved method, it is possibleto get more reliable information about the impact of moisture on shelf life.Thus, one can understand at what initial moisture content the durability is a↵ected. The scientificissue for the study can be summarized as a research question:Can optimization of the Karl Fischer method improve the precision when measuring the initial

moisture content of freeze-dried enzyme beads?

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2 Theory

2.1 Coulometric Karl Fischer (KF) analysis

The basis in determining the water content using coulometric KF is by using the standardreaction equation for the KF reaction.

ROH + SO2 + 3RN + I2 +H2O �! (RNH)SO4R+ 2(RNH)I

The iodine in coulometry is generated by electrochemistry in the coulometric cell through anodicoxidation, in the half-reaction given below:

2I� �! I2 + 2e�

Incorporated in the titration cell is the generator cell, also known as generator electrode and innerburette, this is where the iodine is generated. The potential of the sample is measured with adouble pin platinum electrode, which is located near the measuring electrode. It is measuredwith the help of a voltametric technique through the coulometric titration.Figure 1 illustrates a classical coulometric cell. This cell has two parts, the cathode compartmentand the anode compartment and they are separated with a diaphragm.

Figure 1: Basic setup of the columetric KF titrator. [16]

To generate the iodine a current is applied at the generator electrode. An anolyte is usedin the anode compartment and this is vital for the oxidation. The KF electrolyte (the anolyte)

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contains sulfur dioxide, imidazole and iodide salts and the solvent that is used is ethanol ormethanol. Other solvents may be used dependent on the application. Some others are octanol,chloroform, ethylene glycol or hexanol.

The whole electrochemical reaction would not be possible without the cathode which containsthe reagent. The oxidation reaction on the anode is also depending on a reduction reaction onthe cathode. Di↵erent catholytes can be used but they are often either a manufacturer-specificspecial reagent or it can be the same reagent that you have for the anode compartment. InFigure 2, the anode and cathode reactions are illustrated.

Figure 2: The illustration shows the electrochemical reactions. By oxidation of iodide, iodineis created at the anode. These negative iodide ions generate electrons which forms iodine atthe anode, and these will react with water. Then at the cathode hydrogen is produced by thereduction of hydrogen ions, which is the main product. [16]

The anodic reaction is as mentioned above:

2I� �! I2 + 2e�

The cathodic reaction is:2H+ + 2e� �! H2

For the cathodic reaction an addition of ammonium salts is supplied. This is added sincehydrogen production is desired. Free amine and hydrogen are formed when the ammonium ions

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are reduced. At the anode there will also be some methyl sulfonic acid(CH3SO3H) present. Ifthe CH3SO3H reaches the cathode it can lead to reduction to a sulfide compound and this innot desired. [2] [4] [16]

2.2 Generator electrode without diaphragm

The previous sections described a KF titrator with a diaphgram. However, for this optimizationproject a KF titrator without a diaphragm will be used. There are several advantages with usinga generator electrode without a diaphragm such as there is no blockage or contamination of thediaphragm, it is easy to clean, and it has a lower drift.

The generator cell has another geometry to prevent the generated iodine molecules at theanode to directly, at the cathode, reduce to iodide instead of reacting with water. To prevent thisthe cathode is made smaller and is located so that the iodine cannot reach it. Another parameteris that hydrogen gas is generated at the cathode which makes it more di�cult for the iodine tobe reduced to iodide. In Figure 3 the di↵erent setups are demonstrated and the reactions areshown. [7] [16] In Figure 4 the setup of a KF titrator without diaphragm is illustrated. [18]

Figure 3: Illustrations of a titration cell with a generator electrode with and without a diaphragmand the di↵erent reactions. [16]

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Figure 4: A KF titration cell with a current generator without a diaphragm. [16]

2.3 Water determination with KF coulometric titration

The result from KF coulometric titration is obtained by determining the amount of electricalcurrent in Coulomb, C needed for generating iodine. The definition of one coulomb (1 C ) is thequantity of charge transported by an electrical current of one Ampere (A) during one second (s).From this one can get the amount of water. In other words, the basis is that a specific number ofelectrons (N ) is required to reach the end point of an electrolytic titration of water with iodineduring constant-current conditions, and furthermore this takes a certain time.

N =I ⇥ t

F(1)

For the equation above the current is defined as I, t is the time for reaching the end pointand F is Faraday’s constant. [2] For the oxidation reaction one molecule of iodine, I2, two iodideions, are produced, which gives two electrons. These ions will react with water.

2I� �! I2 �! H2O

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Subsequently, for 1 mole of water 2 x 96485 (Faradays constant) C/mol is necessary. Themolar mass of water is 18.015 g/mol and from this it can be obtained that for 1 mg water, 10.712C electrical current requires to be consumed. For an electrochemically optimized KF cell, 100%current conversion e�ciency is assumed. No standardization in needed as time and current canbe accurately obtained. Because of this, coulometry is seen as an absolute method. However, itis important to regularly check the coulometer with a certified water standard.

Current pulses generate the iodine which are pulses of 100, 200, 300 and 400 mA. To regulatethe rate of iodine, the duration, frequency and height of the pulse are varied. For the maximumpulse, height is dependent on the surface and voltage at the generator electrode and as wellthe conductivity of the anolyte. For the two first mentioned, the surface and voltage at thegenerator electrode, it dependent on the kind of coulometer and the conductivity of the anolytefurther it is a↵ected by the additional solvent (hexanol, chloroform, etc.) and the sample. Atstandard conductivity values, the pulses of current will operate at 400 mA. This results in aniodine production rate of the highest 2240 µg water/min. [16]

2.4 Indication - Bipotentiometric indication

For coulometric analysis, bipotentiometric indication is used. In electrochemical terminology;it is also called ”2-electrode potentiometry”. The principle is that to the double pin platinumelectrode a polarization current ”Ipol”, which is a constant AC current is applied. As long aswater reacts with water there is no free iodide in the anolyte solution. To uphold the requiredpolarization current at the electrode a high voltage is necessary (400 mV to 650 mV). Whenthe water in the solution has reacted with the iodine, it will lead to free iodine in the anolytesolution. From here the voltage needs to drop to maintain the polarization current constant,around 50 mV to 100 mV, since the free iodine causes ionic conduction. The titration is thenterminated when the voltage is reduced to a defined value. [16]

2.5 KF titration for samples with very low water content

For samples containing very small amounts of water, an optimized version of KF titration isnecessary. A common problem with sample that are freeze-dried is that the blank value correctionfrom the solvent is too large. This means that the solvent contains more water than the sampleit self and therefore it is not possible to measure the water consent in the sample. Hence, themethod presented in Figure 5 needs to be used.

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Figure 5: Illustration of how the titrated anolyte is obtained from the titration cell, injected inthe septum bottle and sample taken. [16]

A certain volume of the titrated anolyte is removed, which has been titrated to dryness inthe titration cell. To obtain the anolyte a syringe with a long needle is used. The anolyte shouldthen be returned to the titration cell, and the syringe should be rinsed two or three times likethis. The solution is drawn into the syringe and injected into the septum bottle. Following, thesample is incubated, during which the water extraction to occur. Use a syringe and obtain analiquot, this will be injected into the titration cell. But first the syringe should be rinsed with asmall amount of sample. [5] [10] [16]

2.6 Standard Operating Procedure (SOP) used today

Currently at Cepheid, the following procedure is done for measuring the water content in thefreeze dried beads. For each sample, 70-100 mg beads are transferred into 10 ml glass bottles andsealed with rubber stoppers followed by aluminium seals. In each bottle, 3 ml of dry methanolis injected. In the blank sample only 3 ml dry methanol is injected into empty sealed bottles,which are incubated and measured in the same way as the corresponding samples. The sample isplaced 60 min on the table and is being rotated carefully every 15 minutes. After the extraction0.5 ml of sample is obtained and analyzed with the KF titrator. [6] See Appendix 1 for a moredetailed description. The water content in the samples is calculated according to:

Water content (%) =Total amount water in sample

Weight of beads⇥ 100 (2)

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3 Other methods for measuring the moisture content inlyophilized substances

For determination of the water content several other methods can also be used, such as Loss onDrying (LOD), Fourier transform near-infrared spectroscopy (FT-NIR) and thermogravimetry.As mentioned previously KF is one of the most used methods for determination of the watercontent in lyophilized substances but this method requires environment polluting reagents, it istime-consuming and destructive. Therefore one of the above-mentioned methods may be usedsometimes and hence they are discussed in some more detail below. [18]

3.1 Loss on Drying (LOD)

LOD is usually performed under conditions at 105�C and is a method that measures the loss ofweight during these circumstances. The method is suitable for samples that are abundant andwill not melt or decompose at 110�C. During a specific temperature range the drying processtakes place in an oven, in vacuo or in a desiccator. The sample is dried to a constant mass or fora certain time. Additionally, thermogravimetric analysis can be used to determine LOD values.

However, there are some disadvantages with LOD. During the drying process the mass loss isnot only from the water. There are several other components that may evaporate and decompose.Also, another complication is the amount of water that is bound in di↵erent forms to the sub-stance. Furthermore, the method often requires a long drying process of several hours. [1] [9] [13]

3.2 Fourier transform near-infrared (FT-NIR) spectroscopy

At certain times, when a non-destructive and quick method needs to be used, FT-NIR is bene-ficial. In FT-NIR, no reagents are used and it allows a nondestructive and noninvasive analysis.Performing this technique requires minimal preparation and can be done in situ via plastic orglass containers. FT-NIR is sensitive to hydroxyl groups and to hydrogen bonding caused byresidual moisture which makes it suitable for this purpose. [15] In the NIR region, water isstrongly absorbing, and shows di↵erent absorption maxima around 1940, 1450, 1190 and 970nm. This type of water absorption in the NIR region has been well examined owing to the greatimportance of water in the applications of this technique. [13] [14] [18]

4 Methodology

Cepheid has chosen to use coulometric KF titration withouth a diaphragm because of severalreasons. The main motivation for using the KF without a diaphgragm is because of economicalaspects and its ease of use. Samples where also sent to RISE Research Institutes of Sweden were0.5-1 g sample were heated in oven at 130�C and passed for 30 minutes with dry air over to acoulometric Karl-Fischer titration. The results provided from RISE lead Cepheid to chose tooptimize the coulometric KF method.

5 Important factors for optimization of the KF method

When it comes to water determination it is especially hard compared to determining othercompounds since the sample can easily become contaminated by atmospheric moisture and thisis a di�cult factor to eliminate. Contamination is particularly important to handle when it

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comes to samples with extremely low water content such as this case and also for hygroscopicsamples.

5.1 How to work with the sample

When working with a sample with low water content one should preferably access the substanceby opening the sample as little as possible or via a syringe through a septum. Furthermore, sincethere can already be adsorbed water on the walls in the syringe it is of importance to, two orthree times, fill the syringe and discard the substance outside the sample vessel, and continue toflush it repeatedly and here holding the needle inside. Make sure to always wipe o↵ the needlewith a dry tissue before putting it back in the septum. [5] [8]

5.2 Previous research on optimization

Some research has been done in the field of optimizing the KF method with di↵erent findings.Study made on determining the water content in biodiesel discovered that parameters as changingthe extraction time and the dynamic field did not show any satisfactory response to the accuracyand amount of titrated water. However, the mass of the samples seemed to have an impact onthe obtained results on the water content . [17]

Another study made on honeybee pollen, which also has a relatively low water content,showed that sample weight, particle size, titration solvent and testing temperature are factorsthat a↵ected the results and should be optimized. When increasing the temperature, the averagereaction time was reduced. [11] However, from another report made on bee pollen, they obtainedsimilar results but their results also showed a higher water content at higher temperatures. [3]When they investigated the sample weight it showed that in this case that the weight did nota↵ect the results of water content that much.

So, it seems that depending of what type of sample decided to analyze the parameters varies,since in the report regarding the biodiesel the weight had an impact on the results. Furthermore,both the reports on bee pollen provided information that the particle size was a factor to controlthe water content. But one can also understand from this that it is also dependent on what typeof sample that is analyzed what type of electrostatic interactions with water it will have.

From literature studies on the KF titration method for water determination, informationfrom the manual and knowledge from previous studies made at Cepheid one can understand thatare di↵erent parameters that could be optimized. Important factors that need to be controlledor optimized are: the extraction/incubation process, reagents, reaction time, and whether thesample should be dissolved or not.

6 Experimental

6.1 Method

For the optimization process several di↵erent experiments were conducted to optimize all thevarious parameters that may have an impact on the precision and accuracy of the moisturemeasurements.

6.1.1 Mapping of the Grant Scientific Digital Ultrasonic Bath XUB5

A prototype of a sample holder, see Figure 6, was manufactured to evaluate specific spots in theultrasonic bath. Nineteen spots at three di↵erent depths were tested. The size of the holder was25 x 15 cm and the diameter of the holes approximately 2.4 cm.

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Figure 6: Illustration of the designed sample holder.

SonoCheck vials from PEREG GmbH was used as indicators and they work such as they shiftcolour from green to yellow when the ultrasonic energy is su�cient for the shift. In Figure 7 thecolour shifts are shown.

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Figure 7: SonoCheck changes colour from green to yellow when subjected to ultrasonic energy.

SonoCheck vials were placed at position: 1, 2, 3, 4, 5, 6 and 7 at the depth of: 12.5, 8.5 and4.5 cm from the top of the bath. Each sample was first sonicated for 5 minutes and checked andthis was repeated after 5 more minutes. See Figure 8 for example on how the SonoCheck wasplaced in the sample holder.

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Figure 8: SonoCheck placement at di↵erent spots in the ultrasonic bath.

Furthermore positions A, B, C, D, E, F, G, H, I, J, K and L were tested at 4.5 cm from thetop of the bath. Between every run the water was changed and with the same temperature fromthe tap. For every run the bath was degassed for 30 min, temperature set to 25°C and power to100%.

When all the spots had been evaluated, plastic tubes were placed in the holes with the heightof 4.5 cm. The plastic tubes were placed there so that samples easily could be placed in theultrasonic bath. Figure 9 shows the final prototype of the sample holder.

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Figure 9: The final prototype of the constructed sample holder for the Grant Scientific DigitalUltrasonic Bath XUB5. The holder was made from a foam rubber plate and plastic tubes. Holeswere made in the plate were the tubes were placed.

6.1.2 Determination of extraction method

Three di↵erent methods for extracting the water from the beads were evaluated. For this exper-iment dry methanol was not used. Instead of using dry methanol pre-titrated anolyte was usedas the solvent for the extraction process, as presented in section 2.5. CombiCoulomat fritlessfrom Merck was used. The first method was according to the SOP which is described in section2.6. The second, to investigate if the sample could be placed in an ultrasonic bath to enhanceand speed up the extraction process. Lastly, the third was using a shaking incubator.

All samples using the three di↵erent methods were prepared in the same way, as the SOP, butwith di↵erent extraction methods and the water content was calculated according to Equation2.

The extraction processes were:

1. SOP

2. Using the designed sample holder and placing it in the Grant Scientific Digital UltrasonicBath XUB5 with the settings: power 100%, 25°C, sonication time 10 min and 30 mindegassing.

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3. Using the Heidolpd Rotamax 120 shaking incubator at 150 rpm. The samples were placedon the shaking incubator for 10, 20, 30, 60 and 120 minutes

As reference beads were sonicated until destroyed, it was assumed that all the water wouldbe extracted from them. Two di↵erent types of beads were tested: HIV VL TSR and MTB EZR.

6.1.3 Determination of extraction time on the shaking incubator

To determine the optimal extraction time on the shaking incubator tests were performed on threedi↵erent beads and during the conditions and settings listed below:

1. 2, 4, 6, 8, 10, 20 , 30 and 60 min on the shaking incubator at 150 rpm

2. Sonication in the ultrasonic bath until completely destroyed at the settings: power 100%,30°C, sonication 15 min and 30 min degassing

The samples were prepared as described under section 2.6 and the water content calculatedaccording to Equation 2. However, the solvent was pre-titrated anolyte CombiCoulomat Fritlessfrom Merck. The samples sonicated until completely destroyed were used as reference. For everymethod and bead, 5 replicates were tested. The beads used for this experiment were HIV VLTSR, MTB EZR and HIV VL EZR. The HIV VL EZR beads were placed in around 10%RH forapproximately 144 h to gain a higher water content in the beads. The HIV VL EZR beads haddesiccants inside of the bottle they were stored in, therefore they were very dry.

6.1.4 Finding the optimal KF reagent

Two di↵erent reagents were tested and compared: Hydranal-Coulomat AG from Fluka andCombiCoulomat fritless from Merck. Ten replicates from three di↵erent bead types were testedfor each of the anolytes and the samples were prepared as in of section 2.6 and the water contentcalculated according to Equation 2. However, as mentioned before, pre-titrated anolyte was usedinstead of dry methanol. The samples were incubated on the shaking incubator for 30 min at150 rpm. The same beads were used as when determining the extraction time.

6.1.5 Determining the sample/solvent amount

To determine the amount of sample which is optimal, five di↵erent weights were tested. Tenreplicates for each weight was prepared and one type bead was used: MTB EZR. The di↵erentamounts were 35, 70, 100, 200 and 400 mg and the extraction process was 30 min on the shakingincubator at 150 rpm. The samples were prepared as in section 2.6, but with pre-titrated anolyteHydranal-Coulomat AG as solvent, and the water content calculated according to Equation 2.However, the sample containing 400 mg of beads had 5 ml of pre-titrated anolyte added.

6.1.6 Comparison between the old SOP and the optimized KF method with the

help of water standards

Water standard (WS) for coulometric Karl Fischer Titration from Merck with 0.1% water contentwas used. When testing the two di↵erent methods, 10 replicates were done for each and one blank.The used anolyte was Hydranal-Coulomat AG from Fluka.

For the optimized method, approximately 3 ml of WS was transferred into glass bottles andsealed with rubber stoppers followed by aluminium seals for each sample. In each bottle 3 ml ofpre-titrated anolyte was injected. In the blank sample only 3 ml titrated anolyte was injected

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into empty sealed bottles, which was measured in the same way as the corresponding samples.The data were calculated according to Equation 2.

For the old SOP method the same process was conducted but instead of pre-titrated anolytedry methanol was used.

6.1.7 Design of experiments - DOE

A DOE was made in JMP to be able to draw the conclusion if the new optimized method isbetter than the old SOP and which parameters that e↵ects the results of the water contentmeasurements. Two di↵erent parameters were set in the DOE, operator and bead.

Three di↵erent operators tested three di↵erent beads both with the old SOP and the newmethod. The three di↵erent beads contained di↵erent water content: low, medium and high.The beads with low water content were beads that were manufactured in the production asthey normally do. Furthermore, to get beads with higher water content than the normal beadsdi↵erent methods were used.

One method to increase the water content was to dissolve beads in a specific amount of waterand freeze-drying them again them but skipping the secondary freeze-drying cycle. The othermethod was to expose beads in a chamber to humidities. By making saturated salt solutions therelative humidity (%RH) in the chamber could be controlled. Two di↵erent salt solutions weremade: one with lithium chloride and another with potassium acetate. With lithium chloride it ispossible to obtain equilibrium at 11%RH and with potassium acetate higher, around 23%RH. [12]Bead 1 was the beads with high, bead 2 with medium and bead 3 with low water content. InTable 1 the DOE combinations are illustrated.

Table 1: DOE combinations

Operator Bead

1 11 21 32 12 22 33 13 23 3

The operator performed the tests according to SOP as described in section 2.6 and the newmethod. For each bead and method 2 replicates were done. The new method proceeded asfollowed; for each sample approximately 150-200 mg of beads were transferred into 10 ml glassbottles and sealed with rubber stoppers followed by aluminium seals. In each bottle 5 ml of pre-titrated anolyte Hydranal-Coulomat AG was injected. In the blank sample only 5 mL pre-titratedanolyte Hydranal-Coulomat AG was injected into empty sealed bottles, which were incubatedand measured in the same way as the corresponding samples. The samples were placed on theshaking incubator at 150 rpm for 30 min.

20

7 Results

7.1 Mapping of the ultrasonic bath

The results of the levels tested are illustrated in Figures 10, 11 and 12. Holes marked as greenindicate that ultrasonic energy made the SonoCheck shift colour from green to yellow, red meansno shift in colour (too low ultrasonic energy) and if there is more than one colour it indicatesthat the SonoCheck was in the stage of a colour shift. Parts that are white were not tested.

Figure 10: SonoCheck results at the depth of 12,5 cm from the top of the bath


21


When the tests were performed it was observed that a depth of 4.5 cm from the top of thebath gave the best results and therefore more spots were evaluated at that level. Hence, thisdepth was chosen and plastic tubes were cut to that height and placed in the holes to completethe design of the sample holder. However, spot number seven, see Figure 6, was eliminatedbecause of not providing a colour shift at any times.

7.2 Determination of extraction method

Figures 16 and 17 in Appendix 2 illustrate the graphs of the results from the di↵erent extractionmethods. The results from sonicated until completely destroyed was used as the reference (target)as the initial water content in the beads. The reference was used in the 2 sample t-test evaluationfor all of the beads to be able to compare if there is a significant di↵erence between the assumedinitial water content (ref) and the results obtain from the method.

The methods that were eliminated after the experiment were using the sample holder in theultrasonic bath (2 sample t-test, MTB EZR: p=0.048, HIV VL TSR: p=0.358) and the SOP (2sample t-test, MTB EZR: p=0.356, HIV VL TSR: p=0.236). More tests on the shaking incubator(2 sample t-test, MTB EZR:p=0.624, HIV VL TSR: p=0.641) were to be evaluated. Using theshaking incubator provides a more standardized working procedure than using the SOP.

7.3 Determination of extraction time on the shaking incubator

The graphs in Figure 18, 19 and 20 in Appendix 3 presents the water content in the di↵erentbead types during di↵erent extraction conditions and times. From the graphs one can observethat from 2 min to 20 min on the shaking incubator, for all the samples, the water content isincreasing. However, at around 30 minutes this seems to level out. The results from placingthe sample on the shaking incubator from 30, 60 min and sonicate the sample until completelydestroyed gives approximately the same results. When performing statistical evaluations, One-Way ANOVA test, there is no significant di↵erence in the means between the results after 20 minon the shaking incubator and sonicated until completely destroyed which is used as the reference.Table 2, 3 and 4 presents which means that di↵er from each other. In the tables it can be seenin the left column the sample and on the right which other samples it di↵ers from. Therefore, it

22

was concluded that placing the sample 30 min on the shaking incubator would be the suitableextraction time and method to use.

Table 2: One-Way ANOVA - Which means di↵er for MTB EZR?

Sample (min) Di↵ers from

2 4 6 8 10 20 30 60 REF4 4 8 10 20 30 60 REF6 2 10 20 30 60 REF8 2 4 20 30 60 REF10 2 4 620 2 4 6 830 2 4 6 860 2 4 6 8REF 2 4 6 8

Table 3: One-Way ANOVA - Which means di↵er for HIV VL TSR?


2 4 6 8 10 20 30 60 REF4 2 6 8 10 20 30 60 REF6 2 6 8 10 20 30 60 REF8 2 6 20 30 60 REF10 2 6 20 30 60 REF20 2 4 6 8 1030 2 4 6 8 1060 2 4 6 8 10REF 2 4 6 8 10

Table 4: One-Way ANOVA - Which means di↵er for HIV VL EZR?


2 4 6 8 10 20 30 60 REF4 2 6 8 10 20 30 60 REF6 2 4 8 10 20 30 60 REF8 2 4 6 10 20 30 60 REF10 2 4 6 8 20 30 60 REF20 2 4 6 8 1030 2 4 6 8 1060 2 4 6 8 10REF 2 4 6 8 10

7.4 Finding the optimal KF anolyte

In Figure 21 in Appendix 4 the graph of the results from the tested beads and anolytes ispresented. Three di↵erent beads and two anolytes were evaluated for each bead batch. From

23

the graph one can tell that the results look very similar for the both reagents. There was nosignificant di↵erence between the two anolytes, when looking at the standard deviation, for anyof the three di↵erent beads (2-sample standard deviation test, HIV EZR: p=0.705, MTB EZR:p=0.199 HIV VL TSR: p=0.763). A di↵erence that was noticed when performing the tests wasthat Hydranal-Coulomat AG performed the titration faster and often at the same time betweenevery replicate, for the CombiCoulomat fritless there was more variation. Since the resultsindicate that there is no major di↵erence between the reagents the Hydranal-Coulomat AG waschosen because of its other positive aspects.

7.5 Determining the sample/solvent ratio

In Figure 22 in Appendix 4, the results from using di↵erent amounts of sample are presented.From the graph it can be observed that having too little sample gives very imprecise results anda large variation of the results and having more sample provides better results. The standarddeviation, as presented in Table 5 was the best for the samples with 200 mg and 400 mg of beads.Therefore, it was chosen for further studies to use 150-200 mg of sample for future KF tests.This was chosen since it is easier to work with a sample around 200 mg than having as much as400 mg. Furthermore, regarding the amount of solvent, 5 ml was chosen. The amount of samplewas increased since it is easier to handle with more solvent.

Table 5: Standard Deviation for the di↵erent sample/solvent amounts

Sample (mg) Standard Deviation within the results

400 0,046200 0,046100 0,05570 0,10935 0,226

7.6 Comparison between the old SOP and the optimized KF methodwith the help of water standards

It is clear from the results presented in Figure 13 that the old method is not suitable for samplescontaining a water content around 0.1%. To be able to determine if the new method is betterthan the old a water standard with higher water content should be tested. For the old methodthe blank value correction is too large and therefore not suitable.

24

Figure 13: The means of the results when comparing the new method to the old using a waterstandard containing 0.1% water. It can be observed that the old method provides a negativeresult.

7.7 Design of experiments - DOE

From the results, as presented in Figure 14, from the DOE it can be observed that the resultsprovided from the old SOP almost every time gave a lower water content compared with the newmethod. This was probably due to the high water content in the blank sample. The standarddeviation was also lower for the new method, see Table 6.

Table 6: Standard Deviation for the di↵erent beads with the new and old method

Bead New Old

1 0.031 0.0692 0.025 0.0313 0.019 0.033

25

Figure 14: The water content measured by the di↵erent operators on di↵erent beads with thetwo methods

It was thought that the lower results provided by the old method was because of the largeblank value correction from the dry methanol. To be able to confirm this the results from theblank was plotted next to the results from the sample, see Figure 15. From the graph it waspossible to see if the confidence intervals overlap. As seen for the old method they did andtherefore it resulted in giving results that were a bit lower than expected.

26

Figure 15: The blank values plotted next to the sample results. From the graph it can beobserved that for the new method the confidence intervals did not overlap but they did for theold

When doing an ordinary least squares (OLS) analysis on the DOE results it was found thatthe operators did not provide a significant di↵erence. However, the method had a significantimpact when comparing them, see Table 7. An OLS was made to be able to understand theinfluence of various independent variables on the dependency variable, in this case how themethod, operator and bead a↵ect the moisture. Furthermore, the R-value from the analysis(R=0,992879) explained how much of the variance of our dependent variable can be explainedby the independent variable and in our case we have a good degree of fit.

Table 7: p-values from performing OLS

p-value

Bead 6.49 x 10�36

Method 0.0008Operator 0.1821

8 Discussion

The initial idea was that the ultrasonic bath would be a good method for sample preparation.From preliminary experience it had shown that the bath could enhance and fasten the extraction

27

of water from the beads. The plan was to build a sample holder made of plastic tubes and a foamplate, where the glass bottles with sample could be placed. Hence, di↵erent spots in the ultrasonicbath were evaluated to determine the energy distribution in the bath. But, when thereaftercomparing di↵erent extraction methods with the design sample holder in the ultrasonic bath theresults were not as expected. It was observed that the ultrasonic energy was suppressed whentraveling though the plastic walls probably due to that plastic tubes were too thick. Consequently,the method was not chosen to be used as a extraction method.

A new method for extraction was then evaluated, which involved placing the sample on ashaking incubator. The results indicated that this method was su�cient for extraction of thewater in the beads and that placing the sample 20 min on the shaking incubator was enough.However, 30 min on the shaking incubator was decided just to assure a robust extraction. Sta-tistical analysis indicated that the SOP and the shaking incubator were both good methods forextracting the water, but using the shaking incubator provides a better and more standardizedworking flow hence it was chosen. It was also possible to shorten the extraction time from 60minutes to 30 minutes with the shaking incubator.

Since the water contents were unknown in the beads, a reference was necessary. Therefore,samples prepared in the same way as the method tested were placed in the ultrasonic bath untildestroyed. An assumption was made here, that when the beads were completely destroyed allthe water inside would get extracted. Obviously, this does not have to be the case. There couldbe di↵erent phases in the bead, crystalline and amorphous which water could be bonded to invarious ways.

When evaluating the two anolytes used in the KF titration, both of them were tested underthe same conditions and the conclusion was drawn to use the Hydranal-Coulmat AG. This anolytewas chosen because of the titration time which was very even between each run and often muchfaster. Additionally, this notion was supported by the manufacturer of C30S Compact KarlFischer-coulometer, Mettler Toledo (Patrik Osterman, personal communication). However, theanolytes should provide similar results since they are both made for coulometric KF titration.To further compare the two anolytes, water standards can be used to gain more precise resultsto see if there is any di↵erence in accuracy between them.

Regarding the sample/anolyte ratio the same conlusions were drawn by Mettler Toledo. Itwas confirmed that using a large amount of sample gives better results. Furthermore, since itwas decided to increase the amount of sample, more solvent was also necessary. Using 400 mgwas tested and it would be possible with this amount, but it was considered to be inflexible touse that much in such a small glass bottle. Hence, 150-200 mg was selected because it providedgood results and it was convenient to work with that amount.

By using a WS it to be possible to compare the old with the new method. However, the resultsindicated that the blank value from the old method was too large and the water content in theWS analyzed with that method was not possible to obtain. For the old method dry methanolwas used as the solvent and this should only contain 0.003% water. But, the dry methanolcontained more water. This was probably due to the previous handling of the dry methanol.The operators in the production probably opens the bottle several times so it gets contaminatedby atmospheric moisture. Since it was realized that the dry methanol was not so dry it providedmore confidence in why the old method is not good.

The results obtained from the DOE provided the possibility to draw the conclusion that thereis a significant di↵erence between the two methods. Additionally, it can be understood that thereis not a significant di↵erence between di↵erent operators when using the two di↵erent methods.The two the methods are robust against the influence of the operators handling. However, morereplicates should be done to get a higher statistical reliability. In the production department tworeplicates are obtained from each bead type and therefore this was chosen for the experiment.

28

To be able to confirm that if the di↵erence between the old and new method is in favour of thenew method a sample with a known water content should be evaluated.

9 Conclusion

The new optimized method will proceed as follows. For each sample approximately 150-200mg beads are transferred into 10 ml glass bottles and sealed with rubber stoppers followed byaluminium seals. In each bottle 5 ml of pre-titrated anolyte Hydranal-Coulomat AG is injected.In the blank sample only 5 ml of pre-titrated anolyte Hydranal-Coulomat AG is injected intoempty sealed bottles, which are incubated and measured in the same way as the correspondingsamples. The sample on the Heidolph Rotamax 120 shaking incubator at 150 rpm for 30 minutes.After the extraction 1 ml of the sample is obtained and analyzed with the Mettler Toledo C30SCompact Karl Fischer-coulometer.

There is a significant di↵erence between the new and the old method and it is in favour of thenew method. By understanding that the blank value correction is larger in the old method onecan understand that the old method provides a lower water content then expected. Furthermore,the standard deviation is lower for the new method which indicates lower variation between themeasured samples. Nonetheless, to draw the full conclusion that the new optimized method isbetter than the old a sample with known water content should be analyzed with the two methods.

References

[1] Specification of drug substances and products : development and validation of analytical

methods. Progress in Pharmaceutical and Biomedical Analysis. 2nd ed. edition, 2014.

[2] Victoria C. Dominguez, Cole R. McDonald, Matt Johnson, Doug Schunk, Rod Kreuter, DanSykes, Benjamin T. Wigton, and Balwant S. Chohan. The characterization of a custom-builtcoulometric karl fischer titration apparatus. Journal of Chemical Education, 87(9), 2010.

[3] Iosif Gergen, Florina Radu, Despina Bordean, and Heinz-Dieter Isengard. Determination ofwater content in bee’s pollen samples by karl fischer titration. Food Control, 17(3):176–179,2006.

[4] Honeywell. Hydranal manual eugen scholz reagents for karl fischer titration. http://www.silicol.co.il/web/8888/nsf/web/1145/168608ImageFile3.pdf, February 2019.

[5] Honeywell Fluka. HYDRANAL Laboratory Report L 135, 2019.

[6] Cepheid Inc. Instruktion for karl fischer, fukthalsbestamning av frystorkade beads. Unpub-lished document.

[7] M. Lanz, C.A. de Caro, K. Ruegg, and A. de Agostini. Coulometric karl fischer titrationwith a diaphragm-free cell: cell design and applications. Food chemistry, 96:431–435, 2006.

[8] William Larsson. New approaches to moisture determination in complex matrices based

on the Karl Fischer Reaction in methanolic and non-alcoholic media. PhD thesis, UmeaUniversity, Chemistry, 2008.

[9] K.P. Malik, C Duru, M Ahmed, and Paul Matejtschuk. Analytical options for the measure-ment of residual moisture content in lyophilized biological materials. American Pharmaceu-

tical Review, 13:42–47, 07 2010.

29

[10] Mettler Toledo. Sample Preparation for Karl Fischer Titration, 2019.

[11] Marcelo A. Morgano, Raquel F. Milani, Marcia C.T. Martins, and Delia B. Rodriguez-Amaya. Determination of water content in brazilian honeybee-collected pollen by karl fischertitration. Food Control, 22(10):1604–1608, 2011.

[12] Omega. Equilibrium relative humidity saturated salt solutions. https://www.omega.co.uk/temperature/Z/pdf/z103.pdf, May 2019.

[13] Nilusha L.T. Padivitage, Jonathan P. Smuts, and Daniel W. Armstrong. Chapter 11 - waterdetermination. In Christopher M. Riley, Thomas W. Rosanske, and Shelley R. Rabel Riley,editors, Specification of Drug Substances and Products, pages 223 – 241. Elsevier, Oxford,2014.

[14] Gabriele Reich. Near-infrared spectroscopy and imaging: Basic principles and pharmaceu-tical applications. Advanced Drug Delivery Reviews, 57(8):1109 – 1143, 2005. Non-InvasiveSpectroscopic and Imaging Techniques in Drug Delivery.

[15] Thermo Fischer Scientific. Determination of moisture content in freeze-dried ma-terials by ft-nir spectroscopy. https://assets.thermofisher.com/TFS-Assets/CAD/Application-Notes/D14186~.pdf", 2019.

[16] Mettler Toledo. Good titration practice in karl fischer titration. https://www.mt.com/dam/LabDiv/Campaigns/TestingLabs2013/moisture/package/gtp-karl-fischer-EN.pdf,2019.

[17] M. P Vicentim, M. V Barreto Sousa, V Fernandes Da Silva, V. Lionel Mateus, J. M Ro-drigues, and V Smarcaro Da Cunha. Water content determination in biodiesel: Optimiza-tion of methodology in coulometric karl fischer titration. Journal of ASTM International,7(2):1–7, 2010.

[18] Yiwu Zheng, Xuxin Lai, Susanne Wrang Bruun, Henrik Ipsen, Jørgen Nedergaard Larsen,Henning Løwenstein, Ib Søndergaard, and Susanne Jacobsen. Determination of moisturecontent of lyophilized allergen vaccines by nir spectroscopy. Journal of Pharmaceutical and

Biomedical Analysis, 46(3):592–596, 2008.

10 APPENDICES

10.1 APPENDIX 1

The SOP used today at Cepheid by the operators.

30

Document Number: D25701

Rev: G Effective: 04/25/2019

p. 1 of 6

Instruktion för Karl Fischer, fukthaltsbestämning av frystorkade beads

Cepheid AB Utskrivet dokument är en okontrollerad kopia.

Endast giltigt när dokumentet är kontrollerat mot giltig version i datasystemet och signerat.

Innehållsförteckning Innehållsförteckning ............................................................................................................. 1 1. Syfte ............................................................................................................................. 1 2. Ansvar och befogenheter ............................................................................................. 1 3. Metodhänvisningar ....................................................................................................... 1 4. Definitioner ................................................................................................................... 1 5. Särskild hantering vid användning av KF titrering ........................................................ 2 6. FUKTHALTANALYS AV BEADS .................................................................................. 3

6.1 Kontroll av Karl Fisher Water-Standard 1g/0,1mg (0,01 %) ....................................... 3 6.2 Prov-preparation beads .............................................................................................. 4 6.3 Fukthaltanalys av beads ............................................................................................ 5 6.4 Uträkning och resultat av fukthalt ............................................................................... 6

1. Syfte Syftet med denna instruktionsmetod är att beskriva hur sieving utav beads utförs och vilka visuellt godkända samt underkända kriterier vi har.

2. Ansvar och befogenheter Chef samt L-O ansvarar för att all personal är kvalificerad genom att de har fått erforderlig träning och utbildning. All kvalificerad personal ansvarar själv för att hålla sig uppdaterad samt att använda föreskriven skyddsutrustning.

3. Metodhänvisningar IT0034 Förhållnings- och inträdesregler för arbete i produktionsutrymme på Cepheid AB. KEM0002 Kemikaliregister D25955 SWEDEN - SOP Underhåll av Karl Fisher Coulometer C20 D33510 SWEDEN – WinWedge Instruktion

4. Definitioner • Karl Fisher Coulometer C20 kallas i fortsättningen för KF. • Med vattenstandard avses Water Standard 1g/0,1mg (0,01%).



p. 2 of 6




5. Särskild hantering vid användning av KF titrering

OBS! Vid avtagning av handskar; Undvik att komma i kontakt med handskarnas yttre sida för att kemikalier inte ska överföras till huden.

Coulomat AG

Water-Standard

Metanol Torr

Molecular Sieves

Risk för allvarliga bestående hälsoskador genom långvarig exponering.

Risk för hälsoskador vid långvarig exponering

Risk för mycket allvarliga, bestående hälsoskador genom långvarig exponering

Risk för lungskador vid långvarig exponering.

Symptom vid exponering: Illamående, andningssvårigheter

Symptom för exponering: Magsmärtor, andnöd, avmagring

Symptom vid exponering: Huvudvärk, trötthet. Illamående, yrsel, ögonirritation, synskador. Symptom vid högre halter: Buksmärtor, andnöd, kräkningar

Symptom vid exponering: Irritation i luftvägar, hosta, bröstsmärtor.

Brandfarligt! Förvaras alltid i brandskåp

Giftigt vid förtäring, hudkontakt samt inandning

Får ej hanteras av gravida eller ammande

Skyddsutrustning – skyddskläder, skyddsglasögon, andningsmask vid behov och dubbla handskar. Luftutsuget ska placeras över kemikalien



p. 3 of 6




6. FUKTHALTANALYS AV BEADS

6.1 Kontroll av Karl Fisher Water-Standard 1g/0,1mg (0,01 %) KF titrator C20

Välj program ”PROV” på huvudmenyn.

Använd snapper/LAF-duk för att bryta en Water-Standard ampull.

Skölj en spruta med ca 0,25mL vattenstandard.

Sug upp; 1 mL vattenstandard i sprutan, använd en torkduk för att ta bort eventuellt

spill ifrån sprutan.

Ställ sprutan på vågen, nollställ sedan.

Tryck ”Start sample” när dess text är vit (Testet går inte att starta innan driften är

10µg eller lägre).

När texten ”Add sample” visas, injicera provet försiktigt i titreringskammaren.

Dra tillbaka sprutans pistong och ställ tillbaka sprutan på vågen, tryck ”OK” på

titratorns display för att starta analysen.

Titeringen startar och titratorn frågar efter vikten. Skriv in den vikt (anges i gram) som vågen visar i dess display. Kicka sedan ”OK”. När titreringen är klar visas resultatet.

För att printa ut resultatet klicka på ”Results” och sedan ”Samples”. Välj det prov du vill skriva ut och klicka ”Print”. Spara kvittot för att kunna klistra in i KF protokollet.

OBS! Resultatet ska ligga mellan 90-110ppm Resultatet i ppm finns längst ner på kvittot.



p. 4 of 6




6.2 Prov-preparation beads Prov Blank

Märk upp en liten glasflaska med ”prov”. Märk upp en glasflaska med ”blank” och förslut med kapsylstängare.

Väg flaskan med gummipropp och kapsyl. Dokumentera elektroniskt i uträkningstabell med hjälp av WinWedge.

Tillsätt beads till flaskan, ca 70-100mg Väg flaskan och förslut sedan med en kapsylstängare. Dokumentera elektroniskt i KF protokoll med hjälp av WinWedge.

Tillsätt 3mL metanol. Väg flaskan, dokumentera elektroniskt i KF protokoll med hjälp av WinWedge.

Tillsätt 3mL metanol.

Låt proven extraheras i 60 min. Dokumentera start-och stopptid i KF protokoll.

Blanda provet försiktigt med en roterande rörelse varje kvart.

Extrahering och provtagningen får ej överskrida 90 min!



p. 5 of 6




OBS! Torr metanol till KF-titreringen får endast användas 3 månader efter att flaskan öppnats. Märk upp flaskan med första öppningsdatum. Om för gammal metanol används är det risk för felaktiga svar. Kontrollera alltid detta innan användning!

6.3 Fukthaltanalys av beads KF titrator C20

OBS! Det ska först tas två prover från ”blank” och sedan två prover från ”prov”. Tas proverna av någon anledning i omvänd ordning måste kanyl och spruta bytas ut

mellan ”prov” och ”blank”.

Välj program ”PROV” på huvudmenyn.

Skölj en spruta med ca 0,25mL metanol

Sug upp; 0,5mL metanol i sprutan, torka bort eventuellt spill ifrån sprutan.

Ställ sprutan på vågen, tarera därefter.

Tryck ”Start Sample” när dess text är vit (Då är driften under 10µg).

När texten ”Add sample” visas, injicera provet försiktigt i titreringskammaren.

Dra tillbaka sprutans pistong och ställ tillbaka den på vågen. Tryck ”OK” på titratorns

display.

Analyseringen startar och titratorn frågar efter vikten (anges i gram). Skriv in den vikt som vågen visar i sin display.

När titreringen är klar printas resultatet ut.



p. 6 of 6




Resultatet i anges i ppm och finns längst ner på kvittot.

För in alla värden elektroniskt i KF protokoll och printa sedan ut sidan.

Klistra in resultatkvitton på det utskrivna protokollets baksida.

6.4 Uträkning och resultat av fukthalt Sållas det flera batcher under samma dag i samma rum, kan samma Blank-värden och kalibreringstestresultatet användas för alla prover. Kalibreringsresultatet och Blank-värdena behöver alltså endast tas en gång per dag/rum och beräknas mot alla prover som tas under dagen/rum. Ska Blank-värden och kalibreringstestet användas i flera protokoll behöver resultatet skrivas ut fler gånger. Slutresultatet från ej överskrida 3 % i fukt. Om fallet skulle vara så, kontakta chef för instruktioner.

10.2 APPENDIX 2

Figure 16: MTB EZR beads tested with di↵erent extraction methods

37

Figure 17: HIV VL TSR beads tested with di↵erent extraction methods

38

10.3 APPENDIX 3

Figure 18: The means of the di↵erent water content in HIV VL TSR beads at di↵erent extractiontimes. The green point at 70 min is the reference, sonicated until completely dissolved, whichhas no relation to the time X-axis

39

Figure 19: The means of the di↵erent water content in HIV VL TSR beads at di↵erent extractiontimes. The green point at 70 min is the reference, sonicated until completely dissolved, whichhas no relation to the time X-axis

40

Figure 20: The means of the di↵erent water content in MTB EZR beads at di↵erent extractiontimes. The green point at 70 min is the reference, sonicated until completely dissolved, whichhas no relation to the time X-axis

41

10.4 APPENDIX 4

Figure 21: The di↵erent water contents when using di↵erent pre-titrated anolytes as solvents forextracting water from the beads

42

10.5 APPENDIX 5

Figure 22: The water content in MTB EZR beads when changing the sample/solvent amount.When there is a (2) it means that ratio was tested two times

43

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