NEWSLETTER FROM TOSHVIN ANALYTICAL PVT. LTD.

MAY 2014

•Site Cleaning Validation in Pharmaceutical Manufacturing

• Simultaneous Analysis of Residual Pesticides in Foods via the QuEChERS Method Utilizing GC-MS/MS

• Example of Reflectance Measurement Using Integrating Sphere - Difference Between Diffuse Reflectance and Diffuse + Specular Reflectance Measurements

Electrospray Ionization-Ion Mobility Spectrometry for Rapid On-

TABLE OF CONTENTS

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MAY 20142

Electrospray Ionization-Ion Mobility Spectrometry (ESI-IMS) for Rapid On-siteCleaning Validation (CV) in Pharmaceutical Manufacturing

Abstract

A novel high resolution ion mobility spectrometer (HRIMS) equipped with an electrospray ionization (ESI) source was used to analyze the active pharmaceutical ingredients (APIs) that comprise many of the top selling drugs for use in cleaning validation (CV) of pharmaceutical manufacturing equipment. The ESI-HRIMS system allowed rapid analysis of pharmaceutical samples that were difficult to detect using conventional IMS systems while providing resolving powers, R, greater than 60 and good sensitivity over a greater linear dynamic response range. This research demonstrated analysis of high molecular weight and thermally labile compounds, allowing them to be detected as their intact molecular ions. The high performance IMS system provides a rapid, effective analytical tool that offers improved performance for IMS-based applications.

Introduction

In pharmaceutical manufacturing, equipment contamination can come from any of the materials that have been in contact with the surfaces, including active pharmaceutical ingredients (APIs) from previous runs and cleaning agents. Cleaning validation (CV) is one of the critical control processes in pharmaceutical manufacturing, where equipment must be cleaned before each use, and the cleaning procedure used must be in accordance with good manufacturing practices (GMPs).[1,2] Cleaning validation requires a set of verification procedures that describe specific sampling equipment, techniques and associated analytical methods that are used to demonstrate the efficacy of the cleaning procedure. Companies spend significant resources developing and validating the analytical methods required for cleaning verification. The analytical method must be specific, sensitive, accurate, and precise, and, to be cost-effective, it must be fast and should be easy-to-use.

Companies have several options for effectively sampling and analyzing the residues present on manufacturing equipment. In order to determine the amount of active ingredients in the residue sampled, the pharmaceutical and biotech industries commonly use analytical methods such as high performance liquid chromatography (HPLC) and measurement of total organic carbon (TOC). Under current FDA guidelines for validating cleaning processes, HPLC

analysis of swipe samples collected from the production area is the most commonly used assay. Until the production area is proven clean, the manufacturing process is stopped, waiting for the validation results from analytical laboratories.[3,4] This manufacturing down time results in losses on the order of millions of dollars and the cost is passed on to consumers as part of the pharmaceuticals’ cost.

Other analytical methods are becoming more accepted due to the Process Analytical Technology (PAT) Initiative, including Ion Mobility Spectrometry (IMS). Figure 1 shows the percentage of each analytical method used in 2005. One of the goals of the PAT initiative is to reduce production cycle times by using on-, in-, and/or at-line measurements and controls, and IMS based systems have proven capability for effective operation in non-laboratory conditions.

Currently, two sample acquisition methods are being used for all instruments. Rinse sampling and swab (direct) sampling are commonly used methods for recovering residue from equipment surfaces. The advantages of direct sampling are that the areas that are hardest to clean and which are reasonably accessible can be evaluated. Additionally, residues that are “dried out” or are insoluble can be sampled by physical removal. The two advantages of using rinse sampling are that a larger surface area may be sampled and inaccessible systems or ones that cannot be routinely disassembled can be sampled and evaluated. A disadvantage of rinse samples is that the residue or contaminant may not be soluble or may be physically occluded in the equipment. It is for this reason that the FDA places a good deal of importance on swab sampling, which allows direct sampling of locations that are the toughest to clean and where uneven residue buildup occurs during cleaning. A permutation on swab sampling consists of “direct swabbing & analysis”, whereby the sample is analyzed directly instead of including the

Fig. 1: Percentage of analytical methods used in2005 for cleaning validation.

Other Technologies24%

IMS18%

TOC21%

HPLC37%

MAY 2014 3

intermediate extraction step used in the two current acquisition methods. The IMS method already uses “direct swabbing & analysis” in its primary application of explosives detection. In addition, because the instrument is portable, the cleaning validation can be performed at-line.

The current commercial IMS systems used for cleaning validation were originally designed in the early 1990s for explosives detection and have several drawbacks that have significantly limited the usage of this rapid process analytical technology (PAT). First, the current IMS systems are limited by their quantitation capabilities. Second, because current IMS CV systems use thermal desorption (or GC injector) type sample introduction, only volatile and semi-volatile compounds can be introduced into the spectrometer. Thermally labile compounds, non-volatile compounds and biological samples cannot be analyzed by these commercially available IMS systems. The MW range of the most common pharmaceuticals is between 277 and 586. As the MW becomes greater, the samples are not volatile enough to be introduced into the current IMS systems. Out of the small molecule drugs shown in Table 1, only 5 out of 14 (36%) could be analyzed using the current IMS system configurations. Nevertheless, recent studies using these commercial-off-the-shelf (COTS) systems have proven that the IMS technique worked as well as HPLC for CV, demonstrating the promise of the method. [5,6,7]

Electrospray ionization (ESI) has been shown to allow thermally labile molecules to be ionized as intact ions and analyzed with Excellims’ IMS systems.[8,9] Consequently, Excellims ESI high-resolution IMS (ESI-HRIMS) has been used to study a variety of the APIs comprising the top selling drugs, including many of the high molecular weight and thermally labile compounds. The system has demonstrated improved resolving power, high sensitivity and greater linear dynamic range than the COTS IMS systems for CV using a thermal desorber.

2. Materials and methods

This research was conducted using a commercially available Electrospray Ionization-High Resolution Ion Mobility Spectrometer (ESI-HRIMS) from Excellims Corporation (Acton, MA), as shown in Figure 2. Analyte ions were generated by ESI from liquid samples continuously infused through a 100 µm ID fused silica capillary tube into the ion source at flow rates of 5 µl min-1 using a Chemyx Fusion 100 syringe pump (Stafford, TX). The electrospray needle was held at a potential of 13.4 kV with a current limitation of 1 µA total current. The ionized droplets underwent desolvation in the desolvation region and were subsequently introduced into the drift tube held

at a constant temperature of 150°C via a pulsed Bradbury-Neilson ion gate with gate pulse widths from 40 to 100 µs and a nominal gate voltage of 120 V. The upper potential of the desolvation region of the IMS was held at 8 to10 kV, producing drift fields as high as 850 V cm-1 over the 10.85 cm long drift tube. Ions were separated according to the their mass-to-charge ratio, size and structure as they moved under the influence of the drift field through the 0.8 L/min of counter-flowing drift gas in the drift region as discussed below. The mobility spectrum represented a sum over 10 spectra ranging in length from 10 to 25 ms depending on the drift time of the analyte ions, which were sampled at a Faraday plate detector.

Data were acquired u s i n g E x c e l l i m s

TMVision control and acquisition software and were exported for post-processing to M i c r o s o f t E x c e l . Atmospheric pressure in the laboratory was monitored and reco-rded for all experi-ments to properly correct the drift spectra as shown.

The experiments were performed using high performance liquid chromatography (HPLC) grade solvents, including methanol, water, acetylnitrile and acetic acid, (purchased from Sigma-Aldrich) to dissolve pure chemical standards. Pharmaceutical drugs shown in Table 1 were purchased from Sigma-Aldrich. The drift gas supply used in this project was pure nitrogen (99.999%). Water vapor and other contaminants were removed by passing the gas through a 13X molecular sieve (Fluka) trap before entering the IMS tube.

3. Results and discussion

Representative active pharmaceutical ingredients (APIs) listed in Table 1 were used to detect this group of compounds, determine the system sensitivity and measure the dynamic response range of the ESI-IMS. Known solution concentrations, infusion rate and time were used to calculate instrument sensitivity. For a comparative study, the same samples were loaded on a sample swab of a current commercial IMS cleaning validation system and then placed into the thermal desorber and analyzed according to the instruments’ standard procedure. Small molecule drugs and related compounds were evaluated using ESI-IMS and the commercial IMS system, while large organics and biopharmaceuticals were only studied using the ESI-IMS method.

Fig.2. GA2100 ESI-HR IMS System

MAY 20144

The majority of the compounds in Table 1 are among the top 20 best selling drugs in the US. The selection was made largely dependent on commercial availability of the pharmaceuticals.

Table 1. Pharmaceutical Samples Evaluated

In the current COTS IMS systems designated for CV use, samples are placed into a high temperature thermal desorber at 200-250ºC and the evaporated samples are introduced into a radioactive ionization source. Similar to a GC injector, the thermal desorption method only allows the instrument to analyze samples with low boiling points. For the top selling drugs, only a small percentage of the drug molecules could be analyzed with these instruments. Also, thermal decomposition of labile analyte molecules is commonly observed with current cleaning validation systems. In a conventional thermal desorber, the sample evaporation accompanied with decomposition could generate complex ion mobility spectra, reduce system sensitivity and limit capability for quantitation. In comparison, ESI is cited as a “soft” ionization and sample introduction method; direct liquid sample introduction could result in greater system performance, and in this study, thermally labile compounds did not decompose using ESI. As an example, Figure 3 shows the test result of Irinotecan from the COTS IMS with a thermal desorber. As expected, there is a molecular ion observed and thermal decomposition product ions distributed across the 4-9 millisecond time frame.

Figure 3 clearly shows the drawback of the thermal desorption sample introduction method in this application, i.e. high temperature thermal desorption causes sample decomposition. With this complex fragmentation pattern and poor system resolution, identification of Irinotecan is almost impossible. When Irinotecan is introduced into Excellims’ system via electrospray as shown in Figure 4, two distinguishable peaks were observed. These peaks

+ 2+represent [M+H] and [M+2H] and resemble the test results seen in an electrospray ionization mass spectrometer (ESI-MS) experiment.

Fig. 3 Ion mobility spectrum of Irinotecan produced using COTS IMS with Ni63 ionization and thermal desorption sample

Drift Time (ms)

0Figure 4. Positive ion mobility spectra (under 150 C drift tube condition) of 0.1µg/µl irinotecan HCl in 80/20 MeOH/H O and 0.5% acetic acid 2

Drift Time (ms)

Effexor Venlafaxine 277 Pos

Zoloft Sertraline 306 Pos Yes

Plavix Clopidogrel 321 Pos

Nexium Esomeprazole 345 Pos Yes

Prevacid Lansoprazole 369 Pos/Neg

Norvasc Amlodipine 408 Pos Yes

Risperdal Risperidone 410 Pos

Zocor Simvastatin 418 Pos Yes

Advair Fluticasone 500 Pos Yes

Camptosar Irinotecan 587 Pos Yes

EES Erythromycin 734 Pos

TAXOL Paclitaxel 854 Pos Yes

Vancocin Vancomycin 1485 Pos

Insulin 5808 Pos

Testedon

COTSIMS

TradeName

GenericName

M.W.ExcellimsESI-IMS

Ion Mode

Thermaldecompositionproduct ions fromIrinotecan

MAY 2014 5

Quantitative measurement of trace amounts of chemicals is one of the critical requirements for cleaning validation systems. Figure 5 shows the system response curve from Excellims ESI-IMS system for Risperidone. In a comparative study, the proposed ESI-IMS system demonstrated a linear response range of two orders of magnitude (from 0.001-0.1ug/uL), compared to the only one order of

The zoomed in spectrum in Figure 7 shows the signal-to-noise level when 7.5 picograms (0.001 ug/uL at flow rate of 1 uL/min and signal summing of 450 ms) of risperidone was used to produce this spectra. The noise level in the spectrum is still mainly chemical noise. The true instrument sensitivity could approach sub-picogram level.

Fig. 5 Response/calibration curve for risperidone using Excellims ESI-IMS.

magnitude linear range with the COTS IMS with thermal desorber. Figure 6 shows ion mobility spectra of risperidone obtained using ESI-IMS.

Fig. 6 Ion mobility spectra of risperidone analyzed using Excellims’ ESI-IMS at varying concentration.

Fig. 7 Zoomed-in ion mobility spectrum of risperidone analyzed using Excellims' ESI-IMS at 0.001 µg/ µL

As expected, the thermal desorption based COTS-IMS was effective at detecting small molecules. The ESI-IMS shows similar response to this group of molecules. It is worth noting, however, that most commercially available IMS systems used in cleaning validation have a resolving power of 10-30, where resolving power is commonly defined as R = t /t d 1/2

where t is drift time and t is peak width at half height. d 1/2

With its proprietary design, Excellims’ IMS systems offer R > 60, as shown here.

30

25

20

15

Inte

nsi

ty

10

5

00 0.02 0.04 0.06 0.08 0.10 0.12

y = 2+9.17x + 0.8909Rt = 0.9972

Fig. 8 Ion mobility spectra of Insulin analyzed using Excellims ESI-IMS.

Figure 8 shows an ion mobility spectrum of multiply charged insulin ions. According to previous research under similar electrospray conditions, the predominant ions for the insulin B-chain have 3-5 charges. Intact insulin is known to form dimers and large multimers under broad ranges of solution conditions. The MS/MS studies of the suspected dimer species yield the expected monomers of higher and lower m/z. It is highly unlikely that dimers are formed in the gas phase because of the considerable Coulombic barrier. It is now apparent that such dimeric species were unsuspected contributors to some of the initial MS/MS studies of proteins. In the cleaning validation application, specific peaks are not identified, but the predominant charge state peaks can be used to quantify residual active ingredients.

4. Conclusions

In this study, fourteen active pharmaceutical ingredients (APIs) found in several of the top selling drugs have been analyzed using commercial Electrospray Ionization-High Resolution Ion Mobility Spectrometer (ESI-HRIMS). The observed ion mobility spectra demonstrated that ESI provides a robust way to analyze drug molecules, particularly those with high molecular weight that can’t be volatilized in the COTS IMS systems for CV that employ thermal desorption for sample introduction. ESI also allows thermally labile drug compounds to be ionized in their intact molecular form. IMS with high resolution (R > 60) can provide significantly

[1] ICH Q7A, Good Manufacturing Practices guide for active pharmaceutical ingredients (APIs), 2000.

[2] PIC/S, Guide to Good Manufacturing Practices for Medicinal Products, PE009-9, 2009.

[3] U.S. FDA, Guide to Inspections Validation of Cleaning Processes, 1993.

[4] PIC, Guidance on aspects of cleaning validation in API plants, 2000.

[5] Strege et al., Anal. Chem. 2008, 80, 3040.

[6] Strege, Anal. Chem. 2009, 81, 4576.

[7] Qin et al., J. Pharm. Biomed. Anal. 2010, 51, 107.

[8] Kreuger et al., Int. J. Ion Mob. Spectrom., 2009, 12, 33.

[9] Hilton et al., Accepted to Int J. Mass Spectrom. 2010.

more detail about sample composition, where R is at least a factor of two higher than in the COTS IMS. A linear dynamic response range over two orders of magnitude in concentration has been demonstrated, compared to the one order of magnitude in the current IMS CV instruments. Consequently, an ESI-HRIMS can be used as an effective tool for rapid analysis in cleaning validation applications for a wider range of target pharmaceutical compounds.

References

6 MAY 2014

MAY 2014 7

Simultaneous Analysis of Residual Pesticides inFoods via the QuEChERS Method Utilizing GC-MS/MS

Analytical standards (0.001 mg/L to 0.1 mg/L), as well as samples (0.01 mg/L) created by pretreating paprika with the QuEChERS method and then adding pesticides to the resulting solution, were measured using the analysis conditions shown in Table 1.

ExperimentalThe European Union Reference Laboratory (EURL) has reported their results on evaluating the validity of residual pesticide analysis utilizing GC-MS/MS and LC-MS/MS1). In their report, the measurement of 66 pesiticides using GC-MS/MS was recommended. This data sheet presents selected results of analysis of these pesticides using the triple quadrupole GCMS-TQ8030.

Table 1 Analytical Conditions

GC-MS : GCMS-TQ8030Column : Rxi-5Sil MS (30 m length, 0.25 mm I.D., df=0.25 Dm)Glass Liner : Sky Liner, Splitless Single Taper Gooseneck w/Wool (Restek Corporation, catalog # 567366)[GC]

0Injection Temp. : 250 C0 0 0 0 0Column Oven Temp. : 70 C (2 min) (25 C /min) 150 C (3 C/min) 200 C

0 0(8 C/min) 280 C (10 min)Injection Mode : SplitlessFlow Control Mode : Linear velocity (58.1 cm/sec.)Injection volume : 1 DL

→ [MS]

0Interface Temp. : 250 C0Ion Source Temp. : 230 C

Data Acquisition Mode : MRM (See the below.)

MRM Monitoring m/zCompound Name Quantitative Transition

Precursor>Product CE (V)Qualitative Transition

Precursor>Product CE (V)Compound Name

Precursor>Product CE (V)Quantitative Transition

Precursor>Product CE (V)Qualitative Transition

Diphenylamine 169.10>77.00 26 169.10>115.10 30 Buprofezin 172.10>57.10 18 105.10>104.10 4

Ethoprophos 200.00>157.90 6 200.00>114.00 14 200.00>97.00 26 Bupirimate 273.10>193.20 8 273.10>108.00 18

Chlorpropham 213.10>171.10 6 213.10>127.10 18 beta-Endosulfan 240.90>205.90 14 238.90>203.90 14

Trifluralin 306.10>264.00 8 264.10>206.10 8 264.10>160.10 18 Oxadixyl 163.10>132.10 10 163.10>117.10 24

Dicloran 206.00>176.00 12 206.00>124.00 26 176.00>148.00 12 Ethion 231.00>174.90 14 231.00>128.90 26

Propyzamide 172.90>144.90 16 172.90>109.00 26 Triazophos 161.10>134.10 8 161.10>106.10 14

Chlorothalonil 265.90>230.90 14 265.90>167.90 24 263.90>167.90 24 Endosulfan sulfate 386.90>252.90 10 386.90>216.90 26

Diazinon 304.10>179.10 12 179.20>137.20 18 Propiconazole-1 259.10>190.90 8 259.10>172.90 18 259.10>69.10 12

Pyrimethanil 199.10>184.10 14 199.10>158.10 14 Propiconazole-2 259.10>190.90 8 259.10>172.90 18 259.10>69.10 12

Tefluthrin 197.10>141.10 26 177.10>127.10 32 Tebuconazole 252.10>127.00 24 250.10>125.10 24

Pirimicarb 238.20>166.10 10 166.10>96.00 14 Iprodione 314.10>244.90 12 314.10>56.10 24

Chlorpyrifos-methyl 285.90>270.90 12 285.90>93.00 22 Bromopropylate 340.90>184.90 18 182.90>154.90 16

Vinclozolin 212.10>172.00 14 212.10>144.90 26 212.10>109.00 30 Bifenthrin 181.10>166.10 16 181.10>165.10 22 181.10>153.10 10

Parathion-methyl 263.10>109.00 18 263.10>81.00 26 Fenpropathrin 265.10>210.10 12 181.10>152.10 24 181.10>127.10 26

Tolclofos-methyl 265.00>249.90 12 265.00>93.00 24 Fenazaquin 160.20>145.10 8 145.20>115.10 24 145.20>91.10 24

Metalaxyl 206.20>162.10 8 206.20>132.10 18 Tebufenpyrad 333.20>276.10 8 333.20>171.00 22

Fenitrothion 277.10>125.00 18 277.10>109.00 18 Tetradifon 355.90>158.90 12 353.90>159.00 12 228.90>200.90 14

Pirimiphos-methyl 305.10>290.10 12 290.10>125.00 24 Phosalone 182.00>138.00 8 182.00>111.00 18 182.00>102.10 18

Dichlofluanid 332.00>167.10 6 224.00>123.00 12 Pyriproxyfen 136.10>96.00 12 136.10>78.00 24

Malathion 173.10>117.00 12 173.10>99.00 18 Cyhalothrin 181.10>152.10 24 163.10>127.00 14 163.10>91.00 22

Chlorpyrifos 196.90>168.90 14 196.90>107.00 26 Fenarimol 251.00>139.00 18 139.10>111.00 16

Fenthion 278.10>125.00 22 278.10>109.00 18 Acrinathrin 289.10>93.10 12 181.10>152.10 24 208.10>181.10 8

Parathion 291.10>109.00 14 291.10>81.00 26 Permethrin-1 183.10>168.10 12 183.10>153.10 18 183.10>115.10 24

Tetraconazole 336.10>218.00 18 336.10>204.00 26 Pyridaben 147.20>132.10 14 147.20>117.10 22

Pendimethalin 252.20>162.10 12 252.20>161. 10 12 Permethrin-2 183.10>168.10 12 183.10>153.10 18 183.10>115.10 24

Cyprodinil 225.20>224.10 6 224.20>208.10 18 Cyfluthrin-1 206.10>151.20 24 163.10>127.10 6 163.10>91.00 14

(E)-Chlorfenvinphos 323.10>266.90 14 267.00>159.00 18 Cyfluthrin-2 206.10>151.20 24 163.10>127.10 6 163.10>91.00 14

Tolylfluanid 137.10>91.00 18 137.10>65.00 26 Cyfluthrin-3 206.10>151.20 24 163.10>127.10 6 163.10>91.00 14

Fipronil 367.00>227.90 26 367.00>212.90 26 Cyfluthrin-4 206.10>151.20 24 163.10>127.10 6 163.10>91.00 14

Captan 79.00>77.00 8 79.00>51.00 22 Cypermethrin-1 181.10>152.10 24 163.10>127.10 6 163.10>91.00 14

(Z)-Chlorfenvinphos 323.10>266.90 14 267.00>159.00 18 Cypermethrin-2 181.10>152.10 24 163.10>127.10 6 163.10>91.00 14

Phenthoate 274.10>125.00 18 274.10>121.10 12 Cypermethrin-3 181.10>152.10 24 163.10>127.10 6 163.10>91.00 14

Folpet 147.10>103.10 10 147.10>76.00 26 Cypermethrin-4 181.10>152.10 24 163.10>127.10 6 163.10>91.00 14

Procymidone 283.10>96.10 12 283.10>67.10 24 Ethofenprox 163.20>135.00 10 163.20>107.10 18

Methidathion 145.10>85.00 8 145.10>58.00 18 Fenvalerate-1 125.10>99.00 22 125.10>89.00 22

alpha-Endosulfan 240.90>205.90 14 238.90>203.90 16 tau-Fluvarlinate-1 250.10>200.10 16 250.10>55.00 18

Mepanipyrim 222.20>220.10 8 222.20>193.10 26 Fenvalerate-2 125.10>99.00 22 125.10>89.00 22

Profenofos 337.10>266.80 16 207.90>63.00 26 tau-Fluvarlinate-2 250.10>200.10 16 250.10>55.00 18

Myclobutanil 179.10>152.00 8 179.10>125.00 16 Deltamethrin-1 252.90>93.10 18 181.10>152.10 24

Flusilazole 233.10>165.10 18 233.10>152.10 18 Deltamethrin-2 252.90>93.10 18 181.10>152.10 24

MAY 20148

ResultsCalibration curves for each pesticide obtained by analyzing six calibration standards (0.001 mg/L to 0.1 mg/L), the mass chromatograms for the 0.01 mg/L samples, and the area repeatability (n=6) for each pesticide obtained from the pesticide-spiked samples (0.01 mg/L) are shown below.

Table 2 Area Reproducibility for Each Pesticide (n=6)

Compound Name %RSD

Diphenylamine 4.99 Chlorpyrifos 5.23 Buprofezin 4.92 Fenarimol 5.16

Ethoprophos 4.95 Fenthion 5.75 Bupirimate 5.47 Acrinathrin 2.03

Chlorpropham 6.26 Parathion 6.93 beta-Endosulfan 6.29 Permethrin-1 6.34

Trifluralin 5.33 Tetraconazole 6.96 Oxadixyl 5.74 Pyridaben 7.11

Dicloran 6.49 Pendimethalin 6.29 Ethion 6.18 Permethrin-2 6.24

Propyzamide 5.52 Cyprodinil 5.21 Triazophos 3.45 Cyfluthrin-1 4.44

Chlorothalonil 4.46 (E)-Chlorfenvinphos 5.35 Endosulfan sulfate 4.26 Cyfluthrin-2 3.77

Diazinon 5.45 Tolylfluanid 4.81 Propiconazole-1 6.02 Cyfluthrin-3 7.35

Pyrimethanil 3.18 Fipronil 6.76 Propiconazole-2 5.56 Cyfluthrin-4 8.19

Tefluthrin 5.13 Captan 5.74 Tebuconazole 7.59 Cypermethrin-1 8.58

Pirimicarb 5.00 (Z)-Chlorfenvinphos 5.52 Iprodione 1.72 Cypermethrin-2 3.71

Chlorpyrifos-methyl 5.27 Phenthoate 6.40 Bromopropylate 5.71 Cypermethrin-3 8.08

Vinclozolin 6.33 Folpet 6.56 Bifenthrin 5.29 Cypermethrin-4 2.48

Parathion-methyl 5.81 Procymidone 6.40 Fenpropathrin 4.00 Ethofenprox 5.03

Tolclofos-methyl 4.89 Methidathion 6.17 Fenazaquin 4.84 Fenvalerate-1 4.20

Metalaxyl 5.43 alpha-Endosulfan 6.27 Tebufenpyrad 5.62 tau-Fluvarlinate-1 2.16

Fenitrothion 5.10 Mepanipyrim 6.41 Tetradifon 6.09 Fenvalerate-2 5.65

Pirimiphos-methyl 5.35 Profenofos 5.92 Phosalone 5.90 tau-Fluvarlinate-2 2.14

Dichlofluanid 4.04 Myclobutanil 5.46 Pyriproxyfen 5.16 Deltamethrin-1 7.58

Malathion 6.31 Flusilazole 5.63 Cyhalothrin 5.38 Deltamethrin-2 7.32

Compound Name %RSD Compound Name %RSD Compound Name %RSD

Reference1) EURL-FV Multiresidue Method using QuEChERS followed by GC-QqQ/MS/MS and LC-QqQ/MS/MS for Fruits and Vegetables (European Reference Laboratory, 2010-M1)

MAY 2014 9

Example of Reflectance Measurement Using Integrating Sphere-Difference Between Diffuse Reflectance and Diffuse + Specular

Reflectance Measurements

Reflectance measurements using UV spectrophoto-meters are utilized in a wide variety of fields. The integrating sphere is a spectrophotometer accessory that is particularly effective for analyzing samples that do not have shiny surfaces. Integrating spheres are generally used to measure diffuse reflectance.

Out line of Integrating SphereFig. 1 and 2 show the appearance and structure of integrating sphere.

Integrating sphere is a spectrophotometer accessory effective for analyzing samples whose surfaces are not shiny. All interior surfaces are coated with barium sulfate so that all of the scattered light is focused toward the detector. By changing the angle of incidence, it is possible to switch between diffuse reflectance measurement (incident angle of 0 degrees) and diffuse + specular reflectance measurement (incident angle of 8 degrees). When the angle of incidence is set to 8 degrees, specular reflectance also remains inside the integrating sphere, allowing diffuse + specular reflectance measurement.

In addition, it can be also applied to measure diffuse plus specular reflectance by changing the angle of the light irradiated to the sample. This Application News introduces examples of measuring samples with different surface conditions, using diffuse reflectance, and diffuse + specular reflectance.

Fig.1 Appearance of Integrating Sphere

Photomultiplier (PMT)

Sample Standard white reflectance plate

Fig.2 Schematic diagram of integrating sphere

MAY 201410

Analysis of Plastic Surface

Fig. 3 shows the reflectance spectrum of a plastic surface, obtained with an incident angle of 0degrees (diffuse reflectance). In Fig. 4, the diffuse reflectance spectrum (red line) is overlaid with the spectrum obtained with an incident angle of 8degrees (diffuse + specular reflectance) (blue line). The results clearly show that the reflectivity differs between the two

measurement methods. This is caused by the condition on the plastic surface, where both diffuse and specular reflectance occurs. With the incident angle of 0degrees, only diffuse reflectance is detected. However, by changing the incident angle to 8degrees, both diffuse and specular reflectance is detected simultaneously.

Fig.3 Reflection Spectrum of Plastic Surface (Diffuse Reflectance)

Fig.4 Reflection Spectra of Plastic Surface (Comparison of diffuse reflectance and diffuse + specular reflectance)

Analysis of Tissue Paper

Fig. 5 shows an example of measuring tissue paper. The pink line indicates diffuse + specular reflectance and the green line diffuse reflectance. There is no significant difference between these two spectra. This indicates that suface of tissue paper is rough and almost no specular reflectance occurs. As shown above, by using the integrating sphere and adjusting the incident angle, reflectance spectrum measurements become possible for samples both with and without surface luster.

25.00

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0.00220.0

R%

400.0 600.0 800.0 850.0nm

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R%

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Diffuse reflectance (incident angle: 0º)

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20.00220.0

R%

400.0 600.0 800.0 850.0nm

Flg.5 reflection spectra of tissue paper

Diffuse reflectance + specular reflectance (incident angle: 8º)

Diffuse reflectance (incident angle: 0º)

MAY 2014 11