Characterizing Internet Searchers of Smoking Cessation Information Background The Internet is a viable channel to deliver evidence-based smoking cessation treatment that has the potential to make a large population impact on reducing smoking prevalence . There is high demand for smoking cessation information and support on the Internet . Approximately 7 % -LRB- 10.2 million -RRB- of adult American Internet users have searched for information on quitting smoking . Little is known about these individuals , their smoking status , what type of cessation services they are seeking on the Internet , or how frequently these searches for cessation information are conducted . Introduction The Internet has become the first source of health information for many people , primarily due to the ease of finding information -LSB- 1 -RSB- . In particular , there appears to be great demand for online information and services related to smoking cessation . In a random-digit dial survey conducted in 2004 , 7 % of Internet users in the United States reported using the Web to search for information on `` how to quit smoking '' -LSB- 2 -RSB- ; more women reported to have looked then men -LRB- 10 % vs 7 % -RRB- , and unlike other health-related information seekers , they tended to be younger . At the time , this represented approximately 10.2 million people who had ever turned to the Internet for smoking cessation -- related information or services . Little is known about these individuals , including their basic demographic characteristics , smoking status -LRB- eg , current smokers seeking cessation treatment , recent quitters seeking support to maintain abstinence -RRB- , readiness to quit , quitting history , and treatment preferences . With the proliferation of antismoking sentiments and restrictive smoking policies , a diverse group of individuals may be turning to the Internet for assistance . In order to provide individually tailored and effective cessation treatment services via the Internet , it is necessary to better understand the characteristics and needs of this population . The Internet is a powerful delivery channel that has the potential to deliver behavior change interventions on a population-wide basis to help people modify risk factors such as smoking -LSB- 3 -RSB- . There are limited , but encouraging , data to indicate that Web-based cessation interventions are effective in controlled trials -LSB- 4-6 -RSB- . However , it is not known if these approaches are appealing to or appropriate for the broader population of Internet users seeking cessation assistance . For example , approximately 30 % of visitors to a widely utilized smoking cessation website indicated that they had quit smoking within the past week -LSB- 7 -RSB- . These individuals would be excluded from most randomized clinical trials of smoking cessation treatment , but they may represent a sizable population in need of assistance to remain abstinent . Information and services may need to be specially tailored to address the unique needs of individuals searching for cessation information based on their smoking status , demographic characteristics , and quitting history . The incidence of cessation-related Internet searches may provide an effective proxy for consumer demand for cessation services . To date , there is little information about the rate at which searches for smoking cessation information occur . Several different techniques have been used to estimate the frequency of general health-related Internet searches -LSB- 8-10 -RSB- , with widely varying results . Analyzing the first 300 search terms of the Wordtracker Top 500 keyword list , Phillipov and Phillips found less than 1 % to be health-related terms -LSB- 10 -RSB- . Eysenbach took repeated snapshots of current search terms used on a search engine over a 15-month period , analyzed a random subset of queries , and found that 3.6-5.3 % could be classified as health related -LSB- 8 -RSB- . Fox found that 79 % of surveyed individuals had ever searched for health or medical information , while 7 % had searched for smoking cessation information -LSB- 2 -RSB- . The primary purpose of this study was to characterize individuals who search for smoking cessation information . Specifically , we sought to gather information about sociodemographic and smoking history variables , search patterns -LRB- eg , time of day , search terms used -RRB- , and perceptions about specific types of cessation services . Additionally , we used publicly available data to estimate the incidence of these searches . This information will be critical to develop appropriate and effective online cessation treatment programs , to triage patients as part of a stepped-care treatment model , or to successfully recruit smokers into treatment via the Internet . Methods Recruitment and Eligibility Our recruitment strategy leveraged the prominent position of QuitNet -LRB- www.quitnet.com -RRB- on three of the largest Internet search engines . QuitNet is an established smoking cessation website -LSB- 7 -RSB- that is highly utilized , with over 600000 visitors and 97000 new registrants in 2004 from the United States alone . During the period of this study , it was listed in the top results for queries using `` quit smoking '' or `` stop smoking '' on three large search engines : Google , Yahoo! , and MSN -LRB- Appendix 1 -RRB- . In 2003 , approximately 210000 -LRB- globally -RRB- and 110000 US individuals looking for information on quitting smoking arrived at QuitNet via these search engines . It has been estimated that 80 % or more of Web users seeking health information start from search engines -LSB- 11,12 -RSB- . Research shows that Internet users read search engine results linearly , pay the most attention to the top three to five results , and click on the first promising link they find in the results -LSB- 11,13 -RSB- . Therefore , individuals who click on the link to QuitNet from a search engine results page are likely to be a representative sample of those individuals looking for cessation information on the Internet . We recruited individuals based on four inclusion criteria : -LRB- 1 -RRB- use of the terms `` quit smoking , '' `` quitting smoking , '' `` stop smoking , '' or `` stopping smoking '' in a search engine query ; -LRB- 2 -RRB- use of one of three major search engines -LRB- Google , Yahoo! , or MSN -RRB- to conduct these queries ; -LRB- 3 -RRB- no prior visit to the QuitNet website -LRB- defined as www.quitnet.com or www.quitnet.org -RRB- as determined by the absence of a persistent -LRB- long-term -RRB- tracking cookie ; and -LRB- 4 -RRB- location within the United States as determined by reverse lookup of IP -LRB- Internet protocol -RRB- addresses . When eligible Internet users clicked on the QuitNet link in the results of a search engine query , they were `` intercepted '' and recruited to participate in the study . The recruitment screen contained links to the survey and to the QuitNet website -LRB- Appendix 2 -RRB- . Those who accepted the invitation were directed to the QuitNet website following completion of the survey . Those who declined the survey invitation went directly to the QuitNet website . Recruitment for the survey was conducted for a total of 10 days : it began December 30 , 2003 , was suspended January 1 through January 3 due to technical concerns , and was completed January 12 , 2004 . Generalizabilty was established from the complete panel of respondents , while we restricted further analysis to the respondents that reported any history of smoking and were seeking assistance for themselves . Measures The survey consisted of 10 questions that included basic demographic information -LRB- age , gender -RRB- , reasons for searching for cessation information , current smoking status , readiness to quit , quitting history -LRB- number of past quit attempts , length of quit , quit methods used -RRB- , information desired , and ratings of perceived helpfulness of various online cessation features -LRB- eg , bulletin board , assistance in setting a quit date -RRB- . The survey questions were administered on three separate screens , with no more than three questions per screen . Date and time of survey completion were automatically logged to the database . Data on utilization of QuitNet after survey administration were extracted , including registration and total time online . Time online was defined as the time between the first page view after completion of the survey through the time of the last page view . Statistical Analyses To determine the generalizability of our final sample , we compared survey respondents to nonrespondents who went on to register and use the QuitNet website on the demographic , website utilization , and search pattern variables obtained from the QuitNet database . In addition , we sought to determine the generalizability of our sample to the broader population of individuals who search for online smoking cessation information throughout the year . To do this , we examined the percentage of participants referred from each search engine as well as the total volume of cessation search terms used in Internet search engine queries , using publicly available data from Nielsen/NetRatings -LSB- 14 -RSB- , Overture , and Wordtracker . Chi-square analyses were used to compare our sample to these national datasets . For the 10-item survey , frequency tables were used to summarize the categorical data , and nonparametric tests were used to determine the statistical significance level . We used t tests for normally distributed continuous and ordinal variables . Finally , to estimate the incidence of cessation-related Internet searches each year , we replicated the technique used by Eysenbach and Kohler -LSB- 8 -RSB- . MetaSpy was queried several times per day over the course of 9 months and the active queries were logged . Duplicate results -LRB- defined as the same set of 10 search terms being returned in succession -RRB- were removed . Searches containing the key words `` quit -LSB- ing -RSB- smoking '' or `` stop -LSB- ing -RSB- smoking '' were classified as cessation related . Results Recruitment Outcomes During the 10-day study period , 2265 eligible US residents were intercepted . Of those , 35.8 % -LRB- N = 811 -RRB- clicked on the `` survey '' link , 48 % -LRB- N = 1088 -RRB- clicked on the link to take them directly to the site -LRB- `` declined '' -RRB- , and 16.2 % -LRB- N = 366 -RRB- did neither -LRB- `` abandoned '' -RRB- . Of the 811 individuals who clicked through to the survey , 87.2 % -LRB- N = 655 -RRB- completed the full survey , yielding an overall response rate of 29 % -LRB- Figure 1 -RRB- . Of the survey completers , 29 individuals reported having never smoked , leaving a final sample of 626 respondents . Figure 1 Eligibility and Recruitment Results Generalizability To assess generalizability , we compared all survey participants -LRB- N = 655 -RRB- with nonrespondents who proceeded to register with QuitNet -LRB- N = 243 -RRB- . Overall , nonrespondents -LRB- N = 1454 , abandoned and declined -RRB- were significantly less likely than survey respondents to register on QuitNet -LRB- 16.7 vs 51.4 % , Χ2 2 = 303.7 , P < .001 -RRB- . Compared to survey respondents , nonrespondents spent less time on QuitNet -LRB- 4.5 vs 12.0 minutes , t = 13.4 , P < .001 -RRB- and viewed fewer pages -LRB- 5.9 vs 15.3 pages , t = 16.0 , P < .001 -RRB- on the website . Nonrespondents were more likely to be female -LRB- 59.4 vs 51.9 % , Χ2 2 = 4.2 , P = .02 -RRB- but did not differ by age , smoking status , time of survey invitation , or specific search engine used . As shown in Table 1 , the relative volume of participants referred from each search engine was consistent with national usage patterns -LRB- Χ2 2 = 1.06 , P = .59 -RRB- . In this study , 57 % of participants were referred from Google , 29 % from Yahoo! , and 14 % from MSN . At the time of this study , 60 % of all Internet search queries were estimated to be conducted using Google , 23 % with Yahoo! , and 17 % with MSN -LSB- 14 -RSB- . Table 1 Comparison of search engine usage to Nielsen/NetRatings statistics Relative Reach of Search Engines Search Engine Survey Recruitment -LRB- % -RRB- National Usage -LRB- % -RRB- Google 57 60 Yahoo! 29 23 MSN 14 17 Total 100 100 The use of key search terms -LRB- `` quit smoking , '' `` quitting smoking , '' `` stop smoking , '' or `` stopping smoking '' -RRB- by survey respondents was also consistent with search patterns captured by Overture and Wordtracker . As shown in Table 2 , the most commonly used search term was `` quit smoking , '' which constituted 52.9 % of study queries , 59.1 % of Overture queries , and 47.8 % of Wordtracker queries . `` Stop smoking '' was the second most frequently used search term , which constituted 24.9 % of study queries , 31.1 % of Overture queries , and 36.5 % of Wordtracker queries . Table 2 Frequency of smoking-related search terms in search engine queries Search Term Searches -LRB- % -RRB- Survey Participants -LRB- % -RRB- -LRB- Χ24 = 3.35 , P = .80 -RRB- Overture -LRB- % -RRB- -LRB- Χ24 = 152 , P < .001 -RRB- Wordtracker -LRB- % -RRB- -LRB- Χ24 = 138 , P < .001 -RRB- quit smoking 52.9 55.4 59.1 47.8 stop smoking 24.9 23.9 31.1 36.5 quitting smoking 21.9 20.4 9.0 13.4 stopping smoking 0.3 0.4 0.6 1.8 giving up smoking 0.00 0.00 0.2 0.6 Participant Characteristics As shown in Table 3 , the majority of study participants were female -LRB- 61.2 % , n = 383 -RRB- and between the ages of 26 and 44 years -LRB- 62.7 % , n = 393 -RRB- ; 18.7 % -LRB- n = 117 -RRB- were aged 18-25 years , 17.1 % -LRB- n = 107 -RRB- were aged 45-64 , and less than 1 % were 65 or older -LRB- n = 4 -RRB- or under age 18 -LRB- n = 5 -RRB- . Adjusted to local time of the participant , more than half -LRB- 53.4 % -RRB- of search engine queries for cessation information occurred during work hours -LRB- 8 am-5 pm -RRB- , 26.6 % occurred between 5-9 pm , and 20 % occurred at night -LRB- 9 pm-6 am -RRB- . Participants were asked the reason they were searching for smoking cessation information . The majority of survey respondents -LRB- 90.1 % , n = 590 -RRB- indicated that they were looking for help or support for themselves ; 5.6 % -LRB- n = 37 -RRB- were looking for general information ; 3.4 % -LRB- n = 22 -RRB- were looking for help for someone else ; and 1 % -LRB- n = 6 -RRB- were health professionals or researchers looking for information . Further analyses were limited to individuals looking for cessation help or support for themselves or for general cessation information -LRB- N = 626 -RRB- . Among these individuals , 75.4 % -LRB- n = 472 -RRB- were current smokers , 17.4 % -LRB- n = 109 -RRB- had quit within 7 days -LRB- `` recent quitters '' -RRB- , and 7.2 % -LRB- n = 45 -RRB- had quit more than 7 days ago -LRB- `` longer-term quitters '' -RRB- . Table 3 Demographic and smoking characteristics of study participants -LRB- N = 626 -RRB- Characteristic Number of Participants -LRB- % -RRB- Age < 18 5 -LRB- 0.8 -RRB- 18-25 117 -LRB- 18.7 -RRB- 26-34 232 -LRB- 37.0 -RRB- 35-44 161 -LRB- 25.7 -RRB- 45-54 87 -LRB- 13.9 -RRB- 55-64 20 -LRB- 3.2 -RRB- 65 or older 4 -LRB- 0.6 -RRB- Gender Male 243 -LRB- 38.8 -RRB- Female 383 -LRB- 61.2 -RRB- Smoking Status Current smoker 472 -LRB- 75.4 -RRB- Not thinking of quitting 1 -LRB- 0.2 -RRB- Thinking of quitting in 6 months 222 -LRB- 35.5 -RRB- Thinking of quitting in 30 days 249 -LRB- 39.8 -RRB- Quit ≤ 1 week 109 -LRB- 17.4 -RRB- Quit > 1 week , ≤ 1 month 43 -LRB- 6.9 -RRB- Quit > 1 month 2 -LRB- 0.3 -RRB- The majority of current smokers -LRB- 52.8 % , n = 249 -RRB- planned to quit in the next 30 days , 47.0 % -LRB- n = 222 -RRB- planned to quit in the next 6 months , and one person -LRB- 0.2 % -RRB- was not thinking about quitting . Smokers had made an average of 5.1 quit attempts -LRB- SD = 14.7 ; median = 1 -RRB- during the past year . Information Preferences As shown in Table 4 , information preferences varied by smoking status . Current smokers were more likely than recent quitters and longer-term quitters to be interested in information about how to quit smoking -LRB- 88.1 % , 54.1 % , and 40.0 % , respectively ; Χ2 2 = 104.7 , P < .001 -RRB- and medication usage -LRB- 30.7 % , 5.5 % , and 4.4 % , respectively ; Χ2 2 = 41.0 , P < .001 -RRB- . Not surprisingly , both recent quitters and longer-term quitters were more interested than current smokers in information about withdrawal -LRB- 77.1 % , 66.7 % , and 59.7 % , respectively ; Χ2 2 = 11.7 , P = .003 -RRB- . Table 4 Information sought by smoking status -LRB- N = 626 -RRB- Information Current Smoker -LRB- % -RRB- -LRB- n = 474 -RRB- Quit ≤ 1 Week -LRB- % -RRB- -LRB- n = 109 -RRB- Quit > 1 Week -LRB- % -RRB- -LRB- n = 45 -RRB- Χ22 P value * How to quit 88.1 54.1 40.0 104.7 < .001 Medications 30.7 5.5 4.4 41.0 < .001 Alternative methods 57.6 16.5 17.8 77.3 < .001 Withdrawal 59.7 77.1 66.7 11.7 .003 * Current smokers are the reference group . Note : Multiple responses were allowed , so total percentages within smoking category exceed 100 % . Perceived Helpfulness of Cessation Services Participants were also asked to rate the perceived helpfulness of various smoking cessation treatment interventions on a scale from 1 to 5 , with 1 representing `` very helpful '' and 5 representing `` not helpful at all . '' As shown in Table 5 , the three features that were rated most highly by all participants were -LRB- 1 -RRB- individually tailored information -LRB- mean = 1.90 , SD = 1.18 -RRB- ; -LRB- 2 -RRB- information on withdrawal -LRB- mean = 1.84 , SD = 1.15 -RRB- ; and -LRB- 3 -RRB- a meter that keeps track of personal data -LRB- mean = 2.14 , SD = 1.37 -RRB- . The three features rated the lowest by all participants were -LRB- 1 -RRB- support from a telephone counselor -LRB- mean = 3.21 , SD = 1.35 -RRB- ; -LRB- 2 -RRB- email support -LRB- mean = 2.95 , SD = 1.40 -RRB- ; and -LRB- 3 -RRB- support from others -LRB- mean = 2.90 , SD = 1.38 -RRB- . Ratings of perceived helpfulness varied according to smoking status . Current smokers rated information about medications , assistance in setting a quit date , and assistance in choosing a medication as more helpful than did recent quitters and ex-smokers . Support from others and information about withdrawal received higher ratings of perceived helpfulness from recent quitters and ex-smokers than from current smokers . As detailed in Table 6 , information of withdrawal , individually tailored information , and tracking meters were rated as `` helpful '' or `` very helpful '' by over half of the participants , while telephone counseling was thought to be helpful by less than 30 % of participants . Table 5 Perceived helpfulness of Internet features by smoking status Feature All Participants , Mean -LRB- SD -RRB- -LRB- N = 626 -RRB- Current Smokers , Mean -LRB- SD -RRB- -LRB- n = 472 -RRB- Quit ≤ 1 Week -LRB- n = 109 -RRB- Quit > 1 Week -LRB- n = 45 -RRB- Mean -LRB- SD -RRB- P value * Mean -LRB- SD -RRB- P value * Information on withdrawal 1.84 -LRB- 1.15 -RRB- 1.90 -LRB- 1.17 -RRB- 1.67 -LRB- 1.08 -RRB- .06 1.51 -LRB- 0.75 -RRB- .04 Individually tailored information 1.90 -LRB- 1.18 -RRB- 1.88 -LRB- 1.18 -RRB- 2.00 -LRB- 1.25 -RRB- .36 1.79 -LRB- 0.95 -RRB- .62 A meter that keeps track of personal data 2.14 -LRB- 1.37 -RRB- 2.14 -LRB- 1.37 -RRB- 2.14 -LRB- 1.42 -RRB- 1.0 2.15 -LRB- 1.31 -RRB- .97 Information on medication side effects 2.59 -LRB- 1.38 -RRB- 2.55 -LRB- 1.38 -RRB- 2.79 -LRB- 1.34 -RRB- .11 2.54 -LRB- 1.43 -RRB- .97 Assistance in choosing a medication product 2.72 -LRB- 1.37 -RRB- 2.61 -LRB- 1.36 -RRB- 2.97 -LRB- 1.38 -RRB- .02 3.24 -LRB- 1.24 -RRB- .007 Information on medications 2.72 -LRB- 1.36 -RRB- 2.62 -LRB- 1.36 -RRB- 2.97 -LRB- 1.37 -RRB- .02 3.23 -LRB- 1.23 -RRB- .007 Online , personal help from a professional 2.81 -LRB- 1.38 -RRB- 2.79 -LRB- 1.40 -RRB- 2.86 -LRB- 1.29 -RRB- .67 2.88 -LRB- 1.39 -RRB- .70 Ability to find buddies 2.82 -LRB- 1.37 -RRB- 2.87 -LRB- 1.39 -RRB- 2.74 -LRB- 1.29 -RRB- .40 2.59 -LRB- 1.34 -RRB- .22 Assistance in setting a quit date 2.83 -LRB- 1.39 -RRB- 2.69 -LRB- 1.37 -RRB- 3.25 -LRB- 1.32 -RRB- < .001 3.39 -LRB- 1.37 -RRB- .003 Support via chat , forums , or email 2.90 -LRB- 1.38 -RRB- 2.98 -LRB- 1.39 -RRB- 2.67 -LRB- 1.35 -RRB- .04 2.57 -LRB- 1.30 -RRB- .07 Additional information that arrives by email 2.95 -LRB- 1.40 -RRB- 2.91 -LRB- 1.43 -RRB- 3.06 -LRB- 1.30 -RRB- .34 3.08 -LRB- 1.26 -RRB- .49 Talking by phone with a professional counselor 3.21 -LRB- 1.35 -RRB- 3.17 -LRB- 1.39 -RRB- 3.32 -LRB- 1.22 -RRB- .32 3.46 -LRB- 1.29 -RRB- .20 * P values compared to current smokers ; P = ns for all comparisons between recent and long-term quitters . Note : 1 = very helpful ; 2 = helpful ; 3 = somewhat helpful ; 4 = not very helpful ; 5 = not helpful at all Table 6 Proportion of participants -LRB- N = 626 -RRB- rating Internet cessation services as helpful or very helpful Feature Offered Helpful or Very Helpful n % Information on withdrawal 460 73.5 Individually tailored information 450 71.9 A meter that keeps track of personal data 405 64.7 Information on medication side effects 303 48.4 Information on medications 275 43.9 Assistance in choosing a medication product 273 43.6 Online , personal help from a professional 265 42.3 Ability to find buddies 250 39.9 Assistance in setting a quit date 248 39.6 Support from others , via chat , forums , or email 233 37.2 Additional information that arrives by email 223 35.6 Talking by phone with a professional counselor 184 29.4 Estimating Incidence of Cessation Queries Over the course of 9 months , 541685 searches were extracted from MetaSpy , of which a total of 38 were smoking cessation related . Assuming a total search engine volume of 52 billion searches per year -LSB- 14 -RSB- , this ratio yields an estimate of 3.6 million -LRB- 99 % CI = 2.5-4.8 million -RRB- cessation-related searches per year in the United States alone . Discussion The Internet holds great potential to impact population smoking prevalence by delivering evidence-based treatments to greater numbers of smokers who may never receive treatment through other modalities . This is the first study to characterize the population of individuals looking for cessation information online . Results suggest that the Internet may be an effective way to reach smokers who are younger , who search for cessation services during work hours , and who have recently quit on their own . The relatively large proportion -LRB- 17.4 % -RRB- of recent quitters -LRB- within 7 days -RRB- in this study who are actively seeking assistance is of particular importance . The majority of self-quitters relapse within 8 days -LSB- 15 -RSB- . Over 16 million Americans try to quit on their own each year , but less than 5 % maintain abstinence for 3 months -LSB- 16 -RSB- . Thus , more than 15 million smokers relapse . Until recently -LSB- 17 -RSB- , this segment of the population of smokers received little attention once formal cessation treatments ended . Given the reach and 24/7 availability of the Internet , effective relapse prevention interventions can and should be delivered to the thousands of smokers trying to maintain abstinence . An effective relapse prevention service for self-quitters with intensive support around the quit date could produce a significant impact on smoking prevalence and could be used in conjunction with any other cessation treatment . New population-based strategies to identify and reach smokers with evidence-based cessation treatment are needed -LSB- 3 -RSB- . Currently , telephone quit lines are the primary public health delivery channel for low cost , effective tobacco treatment . Despite the obvious advantages of convenience and cost , uptake rates in states with quit lines have remained low despite aggressive promotion , with less than 2 % of smokers participating -LSB- 18 -RSB- . Given that Internet searchers are more likely to prefer self-help treatment with lower efficacy rates , it is important to design interventions which capture initial interest that can successfully `` up-sell '' more intensive and effective treatment interventions such as telephone counseling and medication use . In this manner , the Internet may be able to provide a workable model for stepped care , where participants can be further triaged to receive telephone counseling ; prescription medication ; in-person , group , or individual counseling ; or even inpatient treatment -LSB- 19 -RSB- . Limitations Several limitations should be considered when interpreting results of this study . The relatively low response rate -LRB- 29 % -RRB- raises concern about the generalizability of findings . Survey respondents were more likely to go on to register with the site ; this likely indicates that they were in a more advanced stage of change than nonrespondents . It may , however , also indicate that the survey itself acted as an incentive to proceed to registration . Furthermore , we worked from the assumption that individuals who clicked on the link to QuitNet in search engine results were representative of the entire population of searchers . Although consistent with utilization patterns of search engines , this assumption has never been tested for searches on smoking cessation , or the QuitNet site in particular . It is possible that less motivated searchers may find the query results unappealing and not click on any link at all , thus biasing our results toward individuals closer to quitting . A second potential limitation is the method we used to estimate the total number of people seeking smoking cessation information each year . This method does not take into account searches using other keywords or individuals using resources other than search engines to find information -LRB- eg , health Web portals , referrals from health professionals , direct-to-consumer advertising , or quit lines -RRB- . In addition , individuals may search for information multiple times , making it difficult to estimate the actual number of unique individuals as opposed to the total number of searches . Finally , the dataset used to derive these estimates is of commercial nature and published online in a promotional context . It has not been peer-reviewed or made available in its raw form . The data for this study were collected from 2003-2004 ; it is possible that in the intervening time the demographics or search behavior of smokers has changed . However , given the limited changes in both search engine technology as well as the demographics of smokers in the United States , this seems unlikely . Despite these limitations , this study provides valuable information about people who search for smoking cessation information online , and it demonstrates a new methodology for validating this kind of survey data . Conclusion This study suggests that the potential public health impact that can be achieved through Internet-based smoking cessation programs is significant given the reach of the Internet -- should these interventions be proven effective . Given that individuals may conduct multiple searches , our estimate of 3.6 million active searches per year for smoking cessation information is consistent with the 2004 data that showed 7 % -LRB- about 10 million -RRB- of Internet users in the United States had searched for information on quitting smoking -LSB- 2 -RSB- . With 1.25 billion smokers throughout the world -LSB- 20 -RSB- , there is enormous potential to globally impact smoking prevalence . The public health community has invested heavily over the past 15 years in successfully de-normalizing smoking and encouraging cessation . However , low uptake rates seen in clinical programs and telephone quit lines call for new population-based approaches . Even if Internet-assisted tobacco interventions prove to have limited efficacy , the Web may still serve as a point of entry to multi-modality treatment programs . These programs may serve to simply link online searchers to more traditional treatment programs -LRB- such as telephone counseling or local group sessions -RRB- , provide pharmaceutical products , or , in more sophisticated settings , use the Web as a platform to integrate voice counseling , local groups , mailed pharmaceutical products , and other proven modalities . We anticipate that the consumer demand demonstrated in this report will ultimately drive increasing services that will reflect a mixture of these different evidence-based treatments .

O B O O B B O O O B O O O O O O O B B O O O O O O O O O O O O O B O O O O O O O B B O O O O O B O O O O O O O O O O O B O O O O O O O B O O O O O O O O O B O O O O O B O O O O O O B O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B B O O O O O O O O O O O O O O O O O O O B O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O O O O O B O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B O O O O O O O O O O O O O O B B O O O O O O B O O O O O O O O O O O O O O O O O O O B B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O B O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O B B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B B O O O O O O O O O O O O O O O O O O B B O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O B O O O O B O O O O B O O O O O B O O O B I B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O B I B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B B O O O O O O O O O O O O O O O O O O B I O O O O O O O B O O O O B B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B I O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O B O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B O O O O O B O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O B B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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Spectrofluorimetric

Determination

of

Oxamniquine

in

Dosage

Forms

and

Spiked

Human

Plasma

through

Derivatization

with

chloride

A

sensitive

simple

and

selective

spectrofluorimetric

method

was

developed

for

the

determination

of

oxamniquine

-LRB-

OXM

-RRB-

in

pharmaceutical

formulations

and

biological

fluids

The

method

is

based

on

the

reaction

between

the

drug

and

chloride

-LRB-

dansyl

chloride

-RRB-

in

presence

of

M

sodium

carbonate

-LRB-

pH

-RRB-

to

yield

a

highly

fluorescent

derivative

that

is

measured

at

nm

after

excitation

at

nm

The

different

experimental

parameters

affecting

the

development

and

stability

of

the

reaction

product

were

carefully

studied

and

optimized

The

fluorescence

concentration

plot

was

rectilinear

over

the

range

of

μg

ml

with

a

lower

detection

limit

-LRB-

LOD

-RRB-

of

μg

ml

and

limit

of

quantitation

-LRB-

LOQ

-RRB-

of

μg

ml

The

proposed

method

was

successfully

applied

to

the

analysis

of

commercial

capsules

The

results

obtained

were

in

good

agreement

with

those

obtained

using

the

official

spectrophotometric

method

Furthermore

the

method

was

applied

for

the

determination

of

oxamniquine

in

spiked

human

plasma

the

mean

recovery

-LRB-

n

-RRB-

is

A

proposal

of

the

reaction

pathway

was

presented

Introduction

Oxamniquine

-LRB-

OXM

-RRB-

-LSB-

-LRB-

isopropylamino

methyl

-RSB-

methanol

is

an

antischistosomal

agent

that

is

indicated

for

the

treatment

of

schistosoma

mansoni

-LRB-

intestinal

schistosomiasis

-RRB-

infection

-LRB-

Fig.

-RRB-

It

has

been

shown

to

inhibit

DNA

RNA

and

protein

synthesis

in

schistosomes

The

oral

bioavailability

of

oxamniquine

is

good

and

effective

plasma

levels

are

achieved

in

h

-LSB-

-RSB-

Fig.

formula

of

oxamniquine

Oxamniquine

is

the

subject

of

a

monograph

in

the

USP

-LRB-

XXIII

-RRB-

-LSB-

-RSB-

whereby

a

spectrophotometric

method

is

recommended

for

its

determination

whether

in

its

pure

form

or

in

capsules

Several

methods

have

been

published

for

the

determination

of

OXM

either

per

se

or

in

pharmaceutical

preparations

and

biological

fluids

These

methods

include

spectrophotometry

-LSB-

-RSB-

non-aqueous

titration

-LSB-

-RSB-

gas

chromatography

-LSB-

-RSB-

HPLC

-LSB-

-RSB-

capillary

electrophoresis

-LSB-

-RSB-

polarography

-LSB-

-RSB-

flow

injection

analysis

-LSB-

-RSB-

cyclic

voltammetry

-LSB-

-RSB-

and

fluorimetry

-LSB-

-RSB-

All

these

methods

are

either

insufficiently

sensitive

or

tedious

and

require

highly

sophisticated

and

dedicated

instrumentation

-LSB-

-RSB-

This

led

us

to

study

the

reaction

of

oxamniquine

with

dansyl

chloride

to

develop

simple

and

sensitive

spectrofluorimetric

method

for

its

determination

in

pharmaceutical

preparations

and

biological

fluids

Dansyl

chloride

is

a

useful

derivatizing

agent

for

primary

amines

secondary

amines

imidazoles

and

phenols

etc.

Several

pharmaceutical

compounds

have

been

determined

through

this

approach

-LSB-

-RSB-

The

reported

spectrofluorimetric

method

for

oxamniquine

-LSB-

-RSB-

is

tedious

and

time

consuming

It

involves

the

use

of

a

hazard

reagent

Experimental

Apparatus

The

spectrofluorimetric

measurements

were

recorded

using

Shimadzu

Spectrofluorometer

equipped

with

Xenon

arc

lamp

Materials

and

reagents

All

reagents

and

solvents

were

of

analytical

reagent

grade

Oxamniquine

pure

sample

was

kindly

provided

by

Pfizer

-LRB-

Sandwich

UK

-RRB-

Capsules

containing

mg

of

Oxamniquine

each

-LRB-

Vansil

capsules

-RRB-

were

obtained

from

commercial

sources

in

the

local

chloride

-LRB-

dansyl

chloride

-RRB-

purchased

from

Sigma

-LRB-

St.

Louis

USA

-RRB-

A

stock

solution

containing

of

dansyl

chloride

was

freshly

prepared

in

acetone

and

was

further

diluted

with

the

same

solvent

to

obtain

solution.Sodium

carbonate

-LRB-

BDH

UK

-RRB-

M

aqueous

solution

-LRB-

pH

-RRB-

Sodium

hydroxide

-LRB-

BDH

UK

-RRB-

M

aqueous

solution.Isobutylmethyl

ketone

-LRB-

IBMK

-RRB-

-LRB-

Merck

Darmstadt

Germany

-RRB-

Plasma

was

kindly

provided

by

Mansoura

University

Hospital

and

kept

frozen

until

assay

after

gentle

thawing.Methanol

and

diethyl

ether

-LRB-

Merck

Darmstadt

Germany

-RRB-

Standard

solutions

A

stock

solution

was

prepared

by

dissolving

mg

of

OXM

in

ml

of

acetone

and

ml

of

M

sodium

carbonate

solution

This

solution

was

further

diluted

with

the

same

solvent

mixture

as

appropriate

The

standard

solutions

were

stable

for

seven

days

when

kept

in

the

refrigerator

General

procedure

Aliquots

of

OXM

standard

solution

were

transferred

into

a

series

of

ml

volumetric

flasks

ml

of

of

dansyl

chloride

reagent

was

added

followed

by

ml

of

acetone

and

mixed

well

The

reaction

mixture

was

left

for

min

and

then

completed

to

the

mark

with

IBMK

the

reaction

mixture

was

allowed

to

stand

for

min

The

fluorescence

intensity

of

the

reaction

product

was

measured

at

nm

after

excitation

at

nm

Blank

experiment

was

carried

out

simultaneously

The

corrected

fluorescence

intensity

was

plotted

vs

the

final

drug

concentration

-LRB-

μg

ml

-RRB-

to

get

the

calibration

graph

Alternatively

the

corresponding

regression

equation

was

derived

Applications

Procedure

for

commercial

capsules

The

content

of

ten

capsules

were

emptied

and

mixed

well

A

weighed

quantity

of

the

powder

equivalent

to

mg

OXM

was

transferred

into

a

small

conical

flask

and

extracted

with

ml

of

methanol

The

extract

was

filtered

into

ml

volumetric

flask

The

conical

flask

was

washed

with

several

milliliters

of

methanol

The

washing

was

passed

into

the

same

volumetric

flask

and

the

combined

extract

was

evaporated

to

dryness

on

a

boiling

water

bath

The

residue

was

dissolved

and

diluted

to

volume

with

a

mixture

of

ml

of

acetone

and

ml

of

M

of

sodium

carbonate

solution

Aliquots

covering

the

working

concentration

range

-LRB-

cited

in

Table

-RRB-

was

transferred

into

ml

volumetric

flasks

The

General

procedure

was

then

applied

The

nominal

content

of

the

capsules

was

determined

either

from

the

calibration

curve

or

using

the

corresponding

regression

equation

Table

data

of

the

proposed

methodParameterProposed

methodConcentration

range

-LRB-

μg

ml

-RRB-

Minimum

detection

limit

LOD

-LRB-

μg

ml

-RRB-

Limit

of

Quantification

LOQ

-LRB-

μg

ml

-RRB-

Correlation

coefficient

-LRB-

r

-RRB-

Sy/x

standard

deviation

of

the

residualsSa

standard

deviation

of

the

intercept

of

regression

lineSb

standard

deviation

of

the

slope

of

regression

line

Error

RSD

n

Procedure

for

spiked

human

plasma

A

stock

solution

containing

μg

ml

of

OXM

was

prepared

Control

samples

of

plasma

was

spiked

with

different

quantities

of

OXM

to

give

a

final

drug

concentration

cited

in

Table

One

molar

NaOH

-LRB-

ml

-RRB-

was

added

to

ml

of

the

spiked

plasma

and

shaken

genteelly

The

solution

was

vortexed

with

ml

of

diethylether

for

min

then

centrifuged

at

rpm

for

min

The

resulting

supernatant

was

evaporated

to

dryness

under

nitrogen

at

ambient

temperature

The

residue

was

dissolved

and

diluted

to

volume

with

a

mixture

of

ml

of

acetone

and

ml

of

M

of

sodium

carbonate

solution

Aliquots

covering

the

working

concentration

range

was

transferred

into

ml

volumetric

flasks

The

recommended

procedures

were

then

applied

The

nominal

content

of

the

drug

was

determined

using

the

corresponding

regression

equation

Results

and

discussion

Dansyl

chloride

was

first

introduced

for

the

determination

of

some

primary

secondary

amines

imidazoles

and

phenols

-LSB-

-RSB-

In

recent

reports

DNS

was

further

used

as

a

fluorogenic

reagent

for

the

determination

of

some

pharmaceutical

compounds

-LSB-

-RSB-

In

the

present

study

OXM

was

found

to

react

with

Dansyl

chloride

at

pH

forming

a

highly

yellow

fluorescent

derivative

with

λ

maximum

emission

at

nm

after

excitation

at

nm

-LRB-

Fig.

-RRB-

Fig.

spectra

of

A

and

B

excitation

and

emission

spectra

of

blank

Fluorescence

spectra

of

A

and

B

excitation

and

emission

spectra

of

oxamniquine

-LRB-

μg

ml

-RRB-

with

dansyl

chloride

at

pH

Optimization

of

experimental

parameters

The

spectrofluorimetric

properties

of

the

colored

product

as

well

as

the

different

experimental

parameters

affecting

the

development

of

the

reaction

product

and

its

stability

were

carefully

studied

and

optimized

Such

factors

were

changed

individually

while

the

others

were

kept

constant

The

factors

include

pH

concentration

of

the

reagent

type

of

buffer

temperature

reaction

time

and

dilution

time

Effect

of

pH

The

influence

of

pH

on

the

fluorescence

intensity

of

the

reaction

product

was

studied

Maximum

fluorescence

intensity

was

obtained

upon

using

mixture

of

acetone

and

M

sodium

carbonate

solution

The

pH

of

the

reaction

mixture

was

found

to

be

pH

was

found

to

be

the

optimum

pH

for

dansylation

because

labeling

of

most

amino

acids

amines

imidazoles

and

phenols

has

been

found

to

be

optimal

at

pH

-LSB-

-RSB-

The

rate

of

dansylation

process

was

found

to

increase

with

increasing

the

pH

value

this

is

due

to

an

increase

in

the

rate

of

hydrolysis

of

dansyl

chloride

into

dansyl

hydroxide

-LSB-

-RSB-

The

latter

shows

strong

fluorescence

and

hence

interferes

seriously

in

the

determination

However

under

the

proposed

chosen

conditions

and

wavelengths

used

there

was

no

interference

arising

from

any

dansyl

hydroxide

formed

as

indicated

by

the

low

fluorescence

intensity

of

the

reagent

Effect

of

concentration

of

dansyl

chloride

The

influence

of

the

concentration

of

dansyl

chloride

was

studied

using

different

volumes

of

of

the

reagent

solution

It

was

found

that

the

reaction

of

dansyl

chloride

with

OXM

started

upon

using

ml

of

the

reagent

in

the

presence

of

sodium

carbonate

-LRB-

pH

-RRB-

Increasing

the

volume

of

the

reagent

produces

a

proportional

increase

in

the

fluorescence

intensity

of

the

reaction

product

up

to

ml

and

remains

constant

up

to

ml

Therefore

ml

of

of

dansyl

chloride

solution

was

chosen

as

the

optimal

volume

of

the

reagent

-LRB-

Fig.

-RRB-

Fig.

of

volume

of

dansyl

chloride

-LRB-

-RRB-

on

the

fluorescence

intensity

of

the

reaction

product

of

oxamniquine

-LRB-

μg

m

-RRB-

at

pH

Effect

of

temperature

Increasing

the

reaction

temperature

higher

than

the

room

temperature

would

result

in

a

subsequent

decrease

in

the

fluorescence

intensity

of

the

reaction

product

Effect

of

reaction

time

Different

time

intervals

were

tested

to

ascertain

the

time

after

which

the

solution

attains

its

highest

fluorescence

intensity

It

was

found

that

after

min

the

reaction

product

reaches

the

highest

fluorescence

intensity

-LRB-

Fig.

-RRB-

and

remains

stable

at

room

temperature

for

min

Fig.

of

reaction

time

of

on

the

fluorescence

intensity

of

the

reaction

product

of

oxamniquine

-LRB-

μg

ml

-RRB-

with

dansyl

chloride

Effect

of

diluting

solvent

Different

solvents

were

tried

to

dilute

the

reaction

mixture

through

out

the

study

It

was

observed

that

isobutyl

methyl

ketone

gave

the

highest

fluorescence

intensity

Dilution

with

M

sodium

carbonate

solution

water

acetone

water

produced

almost

very

week

fluorescence

and

did

not

reduce

the

blank

fluorescence

intensity

While

upon

using

isobutyl

methyl

ketone

the

fluorescence

intensity

attained

its

highest

value

this

was

attributed

to

the

low

fluorescence

value

of

the

reagent

Effect

of

dilution

time

Dilution

times

were

tested

to

ascertain

the

time

after

which

the

solution

attains

its

highest

fluorescence

It

was

found

that

dilution

with

IBMK

after

min

the

reaction

product

reaches

its

highest

fluorescence

intensity

-LRB-

Fig.

-RRB-

Fig.

of

dilution

time

on

the

fluorescence

intensity

of

the

reaction

product

of

oxamniquine

-LRB-

μg

ml

-RRB-

with

dansyl

chloride

Analytical

performance

Validation

of

the

proposed

methods

The

validity

of

the

method

was

tested

regarding

linearity

specificity

accuracy

repeatability

and

precision

according

to

ICH

recommendations

-LSB-

-RSB-

Linearity

By

using

the

above

procedure

linear

regression

equation

was

obtained

The

regression

plots

showed

that

there

was

a

linear

dependence

of

the

fluorescence

intensity

on

the

concentration

of

the

drug

over

the

ranges

cited

in

Table

Linear

regression

analysis

of

the

data

gave

the

following

equation

where

F

is

the

fluorescence

intensity

C

is

the

concentration

of

the

drug

in

μg

ml

and

r

is

the

correlation

coefficient

The

limit

of

quantification

-LRB-

LOQ

-RRB-

was

determined

by

establishing

the

lowest

concentration

that

can

be

measured

according

to

ICH

-LSB-

-RSB-

The

results

are

shown

in

Table

The

limits

of

detection

-LRB-

LOD

-RRB-

were

determined

by

establishing

the

minimum

level

at

which

the

analyte

can

be

reliably

detected

and

the

results

are

also

abridged

in

Table

LOQ

and

LOD

were

calculated

according

to

the

following

equation

-LSB-

-RSB-

Where

σ

is

the

standard

deviation

of

the

intercept

of

regression

line

S

is

the

slope

of

the

calibration

curve

The

proposed

methods

were

evaluated

for

the

accuracy

as

percent

relative

error

-LRB-

Er

-RRB-

and

the

precision

as

percent

relative

standard

deviation

-LRB-

RSD

-RRB-

-LRB-

Tables

and

-RRB-

Table

of

the

proposed

and

official

methods

to

the

determination

of

Oxamniquine

in

pure

formParametersSpectrofluorimetric

methodOfficial

method

-LSB-

-RSB-

No

of

found

-LRB-

-RRB-

Student

's

t

-LRB-

-RRB-

Variance

ratio

F

-LRB-

-RRB-

Figures

between

parentheses

are

the

tabulated

t

and

F

values

respectively

at

p

-LSB-

-RSB-

Accuracy

To

test

the

validity

of

the

proposed

method

it

was

applied

to

the

determination

of

authentic

sample

of

OXM

over

the

working

concentration

range

The

results

obtained

were

in

good

agreement

with

those

obtained

using

official

method

Using

Student

's

t

test

and

variance

ratio

F

test

-LSB-

-RSB-

revealed

no

significant

difference

between

the

performance

of

the

two

methods

regarding

the

accuracy

and

precision

respectively

-LRB-

Table

-RRB-

The

validity

of

the

methods

was

proved

by

statistical

evaluation

of

the

regression

lines

using

the

standard

deviation

of

the

residuals

-LRB-

Sy/x

-RRB-

the

standard

deviation

of

the

intercept

-LRB-

Sa

-RRB-

and

standard

deviation

of

the

slope

-LRB-

Sb

-RRB-

The

results

are

abridged

in

Table

The

small

values

of

the

figures

point

out

to

the

low

scattering

of

the

points

around

the

calibration

line

and

high

precision

Precision

Repeatability

The

repeatability

was

performed

by

applying

the

proposed

methods

for

the

determination

of

two

concentrations

of

OXM

in

pure

form

on

three

successive

times

and

the

results

are

listed

in

Table

Intermediate

precision

It

was

performed

through

repeated

analysis

of

OXM

in

pure

form

using

the

concentrations

showed

in

Table

for

a

period

of

three

successive

days

The

results

are

summarized

in

Table

Table

Validation

of

the

proposed

method

for

the

determination

of

oxamniquine

in

pure

form

Sample

concentration

Recovery

-LRB-

repeatability

-RRB-

Recovery

intermediate

precision

μg

ml

X

SD

RSD

Error

μg

ml

X

SD

RSD

Error

Robustness

of

the

method

The

robustness

of

the

method

adopted

is

demonstrated

by

the

constancy

of

the

fluorescence

intensity

with

the

deliberated

minor

changes

in

the

experimental

parameters

such

as

change

in

the

volume

of

dansyl

chloride

-LRB-

-RRB-

ml

the

change

in

reaction

time

min

and

the

change

in

dilution

time

min

These

minor

changes

that

may

take

place

during

the

experimental

operation

did

n't

affect

the

fluorescence

intensity

of

the

reaction

product

Pharmaceutical

applications

The

proposed

methods

were

then

applied

to

the

determination

of

OXM

in

its

capsules

The

methods

were

tested

for

linearity

specificity

accuracy

repeatability

and

precision

according

to

ICH

recommendations

Specificity

The

specificity

of

the

method

was

investigated

by

observing

any

interference

encountered

from

the

common

capsule

excepients

such

as

talc

lactose

starch

avisil

gelatine

and

magnesium

stearate

These

excepients

did

not

interfere

with

the

proposed

method

Accuracy

The

results

of

the

proposed

methods

were

statistically

compared

with

those

obtained

using

the

official

method

-LSB-

-RSB-

Statistical

analysis

-LSB-

-RSB-

of

the

results

using

Student

's

t

test

and

variance

ratio

F

test

revealed

no

significant

difference

between

the

performance

of

the

proposed

and

reference

methods

regarding

the

accuracy

and

precision

respectively

-LRB-

Table

-RRB-

Table

of

the

proposed

methods

to

the

determination

of

oxamniquine

in

capsulesPreparations

RecoveryProposed

methodOfficial

method

-LSB-

-RSB-

Vansil

capsules

-LRB-

mg

oxamniquine/capsule

-RRB-

Mean

found

-LRB-

-RRB-

SD

t

F

The

tabulated

values

of

t

and

F

are

-LRB-

-RRB-

and

-LRB-

-RRB-

respectively

at

p

-LSB-

-RSB-

Analysis

of

biological

fluid

The

high

sensitivity

of

the

proposed

method

allowed

the

determination

of

OXM

in

spiked

human

plasma

Oxamniquine

is

readily

absorbed

following

oral

ingestion

and

a

peak

concentration

in

plasma

occurs

within

about

h

The

presence

of

food

significantly

delays

absorption

and

limits

the

concentration

achieved

in

plasma

during

the

first

several

hours

after

administration

Urinary

excretion

is

the

major

route

of

elimination

in

man

-LSB-

-RSB-

Oxamniquine

is

given

orally

in

a

dose

of

mg

three

times

daily

this

leads

to

a

final

blood

level

concentration

of

about

μg

ml

i.e.

higher

than

the

upper

limit

of

the

working

concentration

range

of

the

proposed

method

The

high

sensitivity

of

the

proposed

method

allowed

the

determination

of

OXM

in

spiked

human

plasma

The

results

are

shown

in

Table

Table

of

the

proposed

methods

to

the

determination

of

oxamniquine

in

spiked

human

plasmaSampleAmount

added

-LRB-

μg

ml

-RRB-

Amount

found

-LRB-

μg

ml

-RRB-

-LRB-

inter-day

precision

-RRB-

0.020.0195797.850.040.0385696.400.100.0975497.540.200.1985699.28

-LRB-

inter-day

precision

-RRB-

0.200.191495.700.200.196998.450.200.194897.400.200.195597.750.200.197998.950.200.194597.25

Each

result

is

the

average

of

three

separate

determinations

The

extraction

procedure

described

by

Woolhouse

and

Wood

-LSB-

-RSB-

was

adopted

here

The

results

are

satisfactorily

accurate

and

precise

Precision

The

within-day

precision

was

evaluated

through

replicate

analysis

of

Plasma

samples

spiked

with

different

concentrations

of

the

drug

The

percentage

recoveries

based

on

the

average

of

four

separate

determinations

were

thus

indicating

the

high

precision

of

the

method

-LRB-

Table

-RRB-

The

inter-day

precision

was

also

evaluated

through

replicate

analysis

of

plasma

samples

spiked

with

μg

ml

of

drug

on

four

successive

days

The

percentage

recoveries

based

on

the

average

of

four

separate

determinations

were

The

results

are

shown

in

Table

Mechanism

of

the

reaction

The

stoichiometry

of

the

reaction

was

studied

adopting

the

limiting

logarithmic

method

-LSB-

-RSB-

The

fluorescence

intensity

of

the

reaction

product

was

alternatively

measured

in

the

presence

of

excess

of

either

dansyl

chloride

or

OXM

A

plot

of

log

fluorescence

versus

log

-LSB-

dansyl

chloride

-RSB-

and

log

-LSB-

OXM

-RSB-

gave

straight

lines

the

values

of

the

slopes

are

and

respectively

-LRB-

Fig.

-RRB-

Hence

it

is

concluded

that

the

molar

reactivity

of

the

reaction

is

i.e.

the

reaction

proceeds

in

the

ratio

of

Based

on

the

observed

molar

reactivity

of

the

reaction

and

depending

on

the

presence

of

secondary

amino

group

and

by

analogy

to

similar

reports

dealing

with

the

reaction

of

dansyl

chloride

with

compounds

containing

secondary

amino

group

the

reaction

pathway

proposed

in

Scheme

is

presented

Fig.

logarithmic

plots

for

the

molar

ratio

a

Log

F

vs.

Log

-LSB-

dansyl

chloride

-RSB-

b

Log

F

vs.

log

-LSB-

oxamniquine

-RSB-

Scheme

reaction

pathway

between

dansyl

chloride

and

oxamniquine

Conclusion

The

proposed

method

has

the

advantage

of

being

simple

sensitive

and

suitable

for

routine

analysis

in

quality

control

laboratory

Also

it

is

suitable

for

the

determination

of

oxamniquine

in

spiked

human

plasma

with

minimum

detection

limit

lower

than

the

reported

value

In

addition

it

could

be

applied

to

the

determination

of

OXM

in

its

pharmaceutical

preparation

O O O B O B I O B I I O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B I I O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O

oxamniquine
dosage forms
spiked human plasma


Quantitative imaging assay for NF-κB nuclear translocation in primary human macrophages Quantitative measurement of NF-κB nuclear translocation is an important research tool in cellular immunology . Established methodologies have a number of limitations , such as poor sensitivity , high cost or dependence on cell lines . Novel imaging methods to measure nuclear translocation of transcriptionally active components of NF-κB are being used but are also partly limited by the need for specialist imaging equipment or image analysis software . Herein we present a method for quantitative detection of NF-κB rel A nuclear translocation , using immunofluorescence microscopy and the public domain image analysis software ImageJ that can be easily adopted for cellular immunology research without the need for specialist image analysis expertise and at low cost . The method presented here is validated by demonstrating the time course and dose response of NF-κB nuclear translocation in primary human macrophages stimulated with LPS , and by comparison with a commercial NF-κB activation reporter cell line . 1 Introduction Nuclear factor kappa B -LRB- NF-κB -RRB- / rel represent a family of transcription factors , present in all eukaryotic cells , that regulate inducible expression of wide ranging genes involved in immune responses and cell-cycle regulation . Therefore activation of NF-κB provides a key molecular switch that is relevant to many aspects of cellular immunology research . In immune cells NF-κB is most abundant either as a heteromeric complex of two components , p65 -LRB- rel A -RRB- and p50 , or as a p65/p65 homodimer . The p65 component contains the main transactivating domain responsible for NF-κB transcription factor function . Regulation of NF-κB activity is dependent upon cytoplasmic sequestration in association with an inhibitory molecule , IκBα . As a consequence of intracellular kinase signalling cascades IκBα is phosphorylated , and this leads to its degradation , allowing nuclear translocation of p65/rel A and hence so-called `` activation '' of NF-κB -LRB- Ghosh et al. , 1998 -RRB- . Conventional methods for testing NF-κB nuclear translocation utilise a semi-quantitative electromobililty gel-shift assay . This involves incubation of nuclear extracts with 32P-labelled oligonucleotides of NF-κB binding sites and separation from unbound probe by electrophoresis in a non-denaturing polyacrylamide gel . However , this assay is principally limited by sensitivity , and requires large scale cell culture -LRB- typically > 10 × 106 cells -RRB- , thus precluding its use with primary cells . Additional disadvantages are the labour-intensive protocol and the need for radioisotopes . Cell lines transfected with NF-κB promoter -- reporter gene constructs are used widely to study cell signalling pathways , and are easy to quantitate with colorimetry or luminometry , but are not suitable for most primary cell culture studies . Commercial kits for enzyme linked immunosorbent assays of NF-κB quantitation within nuclear extracts have also become available , but their expense may be prohibitive . An attractive new strategy is to image NF-κB translocation from the cytoplasm to the nucleus , using immunofluorescence staining . This can be performed on a small scale and at a single-cell level . Fluorescence microscopy , laser scanning cytometry and flow cytometry to quantify NF-κB nuclear translocation have all been reported -LRB- Deptala et al. , 1998 ; George et al. , 2006 ; Rogers and Fuseler , 2007 ; Fuseler et al. , 2006 -RRB- . Drawbacks of these previous reports are the need for expensive imaging equipment and/or image analysis software . Here we present an alternative simple method for quantitative detection of NF-κB rel A nuclear translocation which uses standard confocal immunofluorescence microscopy and the public domain Java image processing program , ImageJ . We suggest that the general availability of all three components -- fluorescence microscopy , immunostaining reagents , and the analytic protocol-provides a readily accessible method for the study of NF-κB nuclear translocation in primary cell cultures . For this study we have chosen to use a cell system that has been difficult to analyse previously , primary monocyte-derived macrophages that have been isolated from healthy human volunteers . In vivo macrophages are resident tissue mononuclear phagocytic cells derived from circulating monocytes . They function both as sensory cells of innate immunity , and as effectors , initiating early non-specific host defences , both by local recruitment of other immune cells and by induction of an acute phase response , which leads to systemic priming of the immune system . In vitro work on these cells often includes stimulation by model innate stimuli , now known to be Toll-like receptor -LRB- TLR -RRB- ligands , such as lipopolysaccharide -LRB- LPS -RRB- -LRB- TLR-4 -RRB- and Pam3CSK4 -LRB- TLR-2 -RRB- . Activation of the NF-κB pathway is a common downstream component of the cellular response to many different innate immune stimuli and is used frequently in these cells as a biochemical detection and quantification method to study innate immune cellular activation . Therefore analysis of the NF-κB pathway in macrophages , and in related mononuclear phagocytic cells such as dendritic cells , is an important area of research interest . However , analysis in these cells has been hampered by the lack of suitable methods . Myeloid leukaemic cell lines -LRB- e.g. U937 , THP-1 -RRB- are the standard for these experiments , but unless they are very carefully differentiated -LRB- which itself may involve NF-κB activation -RRB- they are not ideal models . This necessity for an accurate and reproducible primary macrophage system stimulated us to adapt the NF-κB assay as described here . 2 Methods 2.1 Macrophage culture and innate immune stimulation Human blood samples were obtained from healthy volunteers . The study was approved by the joint University College London/University College London Hospitals NHS Trust Human Research Ethics Committee and written informed consent was obtained from all participants . Peripheral blood mononuclear cells -LRB- PBMC -RRB- , consisting of monocytes and lymphocytes , were prepared by density-gradient centrifugation of heparinised blood with Lymphoprep ™ -LRB- Axis-Shield -RRB- according to the manufacturer 's instructions . PBMC were resuspended in RPMI 1640 with l-glutamine -LRB- Gibco , Invitrogen -RRB- -LRB- NM -RRB- containing 5 % heat inactivated -LRB- 56 °C for 30 min -RRB- type AB normal human serum -LRB- NHS -RRB- -LRB- Sigma Aldrich -RRB- -LRB- 107 cells / ml -RRB- and seeded on to 13 mm -LRB- No 1.5 -RRB- glass coverslips -LRB- VWR -RRB- using 2 × 105 cells/cover slip . After 1 h at 37 °C non-adherent cells -LRB- lymphocytes -RRB- were removed by sequential washes with Hanks buffered saline solution -LRB- HBSS -RRB- -LRB- Gibco , Invitrogen -RRB- . Adherent cells -LRB- monocytes -RRB- were incubated in NM containing 10 % autologous heat-inactivated HS supplemented with 20 ng/mL macrophage-colony stimulating factor -LRB- M-CSF -RRB- -LRB- R&D systems -RRB- for 3 days . Any remaining non-adherent cells were removed by further washes with HBSS , and NM/HS was replaced , but without additional M-CSF . This protocol yields adherent macrophages by day six with less than 5 % contamination by lymphocytes -LRB- data not shown -RRB- . Ultra-pure LPS -LRB- Invivogen -RRB- and Pam3CSK4 -LRB- Invivogen -RRB- were used as prototypic innate immune stimuli at different concentrations as outlined below . Polymyxin B -LRB- Invivogen -RRB- was used as a specific inhibitor of LPS bioactivity . 2.2 Immunofluorescence staining For immunostaining , rabbit polyclonal affinity purified antibody to rel A -LRB- C-20 -RRB- -LRB- Santa Cruz Biotechnology -RRB- was used -LRB- 2 μg/ml -RRB- with a secondary antibody , Alexa-Fluor -LRB- AF -RRB- 633 -- conjugated F -LRB- ab ' -RRB- 2 goat anti-rabbit IgG -LRB- Invitrogen -RRB- used at 4 μg/ml . 10 % normal goat serum -LRB- Sigma Aldrich -RRB- used as blocking buffer , eliminated all non-specific binding of the secondary antibody -LRB- data not shown -RRB- . Cells cultured on glass coverslips were fixed with 3.7 % paraformaldehyde -LRB- 15 min , room temperature -RRB- and washed with Tris-buffered saline -LRB- TBS -RRB- . Each coverslip was inverted on to 50 μl of solution placed on impermeable Nesco film -LRB- VWR -RRB- . All reagents were diluted in TBS , and coverslips were washed by immersion into TBS between each staining step . Coverslips were incubated sequentially with 0.2 % Triton-X100 -LRB- Sigma -RRB- -LRB- 10 min , room temperature -RRB- , blocking buffer -LRB- 30 min , room temperature -RRB- , primary antibody diluted in blocking buffer -LRB- overnight , 4 °C -RRB- and secondary antibody diluted in blocking buffer -LRB- 1 h , room temperature -RRB- . Nuclei were counterstained with 2 μg/ml of the nuclear stain DAPI -LRB- Sigma Aldrich -RRB- for 5 min . Coverslips were mounted on to glass slides -LRB- VWR -RRB- using Vectashield hard-set mounting media -LRB- Vector -RRB- . 2.3 Image acquisition and analysis Fluorescence images were captured on a Leica SP2 confocal microscope . DAPI -LRB- excitation 405 nm , emission 400 -- 450 nm -RRB- and AF633 -LRB- excitation 633 nm , emission 650 -- 700 nm -RRB- fluorescence were captured using sequential acquisition to give separate image files for each -LRB- Fig. 1A -RRB- . A pin hole of 1 Airy -LRB- 114.5 μm -RRB- , scan speed of 400 Hz and 4 frame averaging was used . Photomultiplier tube gain and offset were adjusted to give sub-saturating fluorescence intensity with optimal signal to noise ratio . Image analysis was performed using ImageJ v3 .91 software -LRB- http://rsb.info.nih.gov/ij -RRB- . Five high power fields were selected for analysis of each stain . To avoid being biased by the NF-κB staining , each field was selected by viewing nuclear -LRB- DAPI -RRB- staining only to identify near-confluent cells and thereby maximise the cell numbers included in the analysis . Preliminary experiments demonstrated that this approach provided data on at least 500 cells , and that the variance of the data was not changed by increasing the sample size further -LRB- data not shown -RRB- . For each high power field , binary image masks were created of rel A and DAPI positive staining to define regions of interest -LRB- ROI -RRB- for analysis . This was done by applying a median filter -LRB- 3 × 3 pixel radius -RRB- to remove noise and to approximate the distribution of staining intensity to a median value -LRB- Fig. 1B -RRB- . Automatic thresholding , using the Isodata algorithm -LRB- Ridler and Calvard , 1978 -RRB- was then used to convert the image to a binary mask -LRB- Fig. 1C -RRB- that included all fluorescence data above background . The DAPI staining mask was used to define the nuclear ROI . Using the image calculator , the DAPI mask was subtracted from the rel A mask to create a staining mask defining the cytoplasmic ROI . Each of these ROI masks were then applied , by the image calculator , to the original rel A -LRB- AF633 -RRB- staining images to separate nuclear and cytoplasmic staining within each high power field -LRB- Fig. 2A -RRB- . Quantitative fluorescence data were exported from ImageJ generated histograms into Microsoft Excel software for further analysis and presentation -LRB- Fig. 2B -RRB- . Measurement of nuclear fluorescence alone does not distinguish NF-κB nuclear translocation from increased background levels of NF-κB expression or artefactual differences in staining intensity . Therefore nuclear and cytoplasmic staining intensities were compared to give the nuclear : cytoplasmic ratio as a relative measure of rel A nuclear localisation . Nuclear and cytoplasmic histogram data were first normalised for the total number of data points included in the analysis and then comparison was made of the sum of staining intensities . In this way , NF-κB nuclear translocation is represented by an increase in nuclear : cytoplasmic ratio of rel A staining . Multiple images can be batch processed simultaneously in this way by converting individual images into DAPI and NF-κB rel A stacks . 3 Data presentation and discussion To validate this methodology and analysis , time course and dose response studies of LPS induced NF-κB nuclear translocation were performed . Macrophages were stimulated with 0 -- 100 ng/ml LPS in NM with 10 % HS for up to 1 h. NF-κB nuclear translocation was evident by 30 min and maximal at 60 min -LRB- Fig. 3 -RRB- . The expected increase in nuclear : cytoplasmic ratio was also evident across the LPS dose range -LRB- Fig. 4 -RRB- . In addition we tested the effect of Polymyxin B -LRB- PMB -RRB- , a polypeptide that binds and neutralises the bioactive lipid A component of LPS . PMB completely abrogated NF-κB nuclear translocation in response to LPS specifically , and had no effect on stimulation with the TLR2 ligand , Pam3CSK4 -LRB- Fig. 5 -RRB- . To further evaluate the quantitation provided by this assay the results were compared with a commercial NF-κB reporter gene assay using the HEK-293 cell line transfected with TLR2 and a secreted alkaline phosphatase NF-κB reporter gene construct -LRB- Invivogen -RRB- . Transfected cells were seeded on to glass coverslips as described above and allowed to adhere for 48 h . They were then stimulated with Pam3CSK4 -LRB- dose range 0 -- 2 μg/ml -RRB- diluted in the manufacturer 's detection media . Cells were fixed and stained as above for immunofluorescence staining of NF-κB after 1 h stimulation . Duplicate wells were allowed to incubate for 6 h to allow the reporter gene and substrate reaction to take place . Cell culture supernatants were then harvested to quantify the colorometric reaction spectrophotometrically at 630 nm . Quantitative comparison of NF-κB activation using the reporter gene expression assay and NF-κB nuclear translocation by confocal microscopy showed statistically significant correlation -LRB- Fig. 6 -RRB- . Importantly , reporter gene expression provides a measure of NF-κB function that is dependent on its nuclear translocation , but not exclusively regulated by it . The difference between measurement of nuclear translocation and NF-κB function is acknowledged and may in part explain the imperfect regression analysis -LRB- r2 0.83 -RRB- between the two methods . The method proposed here is most suitable for adherent cell cultures with relatively large cytoplasmic : nuclear area ratios that allow clear distinction between nuclear and cytoplasmic NF-κB staining . It requires relatively few cells and can be used to study NF-κB nuclear translocation at single-cell level or in mixed cultures . It can be readily applied to the study of NF-κB activation in macrophages , dendritic cells , epithelial and endothelial cells , and fibroblastic cells . We have also been able to apply this method to monocytic cells in suspension -LRB- THP-1 and K562 cell lines -RRB- by air drying them onto coverslips for immunostaining -LRB- data not shown -RRB- , albeit their typically small nuclear : cytoplasmic area ratio may limit the accuracy of quantitation . This generic image analysis methodology may be applied to quantitative analysis of other transcription factors and signalling events in which assessment of sub-cellular localisation is necessary . Where confocal microscopy facilities are available , this method overcomes the problems related to sensitivity , use of radioisotopes and cost . It can be easily adopted in current cellular immunology research , and given the ready accessibility of the public domain image analysis software , with further validation this methodology may serve as a universal standard that allows better comparison of data from separate experiments and different research groups .

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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O 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nuclear factor-kappa b
confocal immunofluoresence