Quality Control of Traditional Chinese Medicine: Analysis of marker components in Banlangen and aflatoxin in Zizyphus fructus by LC/MS

本論文共分為三部分，皆屬中藥材品管之研究，研究之中藥材包括板藍根、大青葉及大棗，所用之分析儀器為液相層析質譜儀(LC/MS)及液相層析串聯質譜儀(LC/MS/MS)，其摘要內容如下: 第一部份主要是利用液相層析質譜儀之大氣壓化學游離法(LC/APCI-MS) 正離子模式來偵測中藥材大青葉(Daqingye)及板藍根(Isatis indigotica, Banlangen)中三個指標成份色胺酮(tryptanthrin)、靛藍(indigo)、靛紅(indirubin)的含量；在本篇研究中，成功地發展出一種快速、具選擇性、及高靈敏性的LC/APCI-MS的方法，用以偵測及分析大青葉及板藍根中的色胺酮、靛藍、及靛紅之含量，此三個指標成份的偵測範圍為100~1500 ng/mL，線性相關係數的平方值大於0.996，精密度的相對標準偏差值(RSD)小於9.5%，且回收率大於86.6%。將此方法應用於真實植物樣品的分析，選擇21個來自不同地區的板藍根樣品，分析其新鮮、乾燥葉、及乾燥根中色胺酮、靛藍、及靛紅的含量，其結果可做為此植物在選種及育種方面的參考。最後，比較液相層析紫外光偵測器(HPLC-UV)的方法，此LC/APCI-MS的方法可以成功地用來分析大青葉及板藍根中的三種指標成份的含量。 第二部份是以液相層析質譜儀之大氣壓化學游離法正離子模式，來偵測中藥材板藍根中之指標成份色胺酮、靛藍、靛紅的含量，所不同的地方在於本部份著眼於分析比較板藍根奈米化顆粒前後，指標成份含量的差異性，其結果為奈米化板藍根藥材(粒徑300 nm) 之三種分析物含量 > 微米化板藍根(粒徑5 µm) > 實驗室的研磨尺寸(~0.5 mm)，其中色胺酮之含量在微奈米化後約增為原來2.4倍，靛藍含量增加3.4倍以上，而靛紅則增加3.3倍，這說明了板藍根藥材在奈米化後，可能由於其總表面積變大，增加了甲醇在單位時間的萃出效果。 對於板藍根科學中藥的測定，因受到大量的基質干擾而無法準確的定量分析，此基質干擾有可能是來自科學中藥中的定型劑，須先了解定型劑的種類為何，再配合前處理步驟，如固相萃取，用以淨化其複雜基質。 第三部分主要是探討利用液相層析串聯質譜儀之大氣壓化學游離法(LC/APCI-MS/MS)正離子模式來對中藥材大棗(Zizyphi jujube, Ziphus fructus)之黃麴毒素作定量分析。本部份以超臨界流體萃取(supercritical fluid extraction, SFE)技術配合LC/APCI-MS/MS的分析方法，可成功的應用在傳統中藥大棗中黃麴毒素B1，B2，G1，G2 (AFB1，B2，G1，G2)的測定。為了減少大棗中複雜基質的干擾，須利用超臨界流體萃取技術的高選擇性特點配合LC/MS/MS測定，檢測奈克級的黃麴毒素。游離化的模式包含電灑游離法(electrospray ionization, ESI)以及大氣壓化學游離法(atmosphere pressure chemical ionization, APCI)，並比較二者間的偵測效率，以達到最佳化的質譜測定條件。AFB1，B2，G1，G2的校正曲線濃度在介於1 ~ 50 ng/g 時呈線性，其線性相關係數的平方值(r2)大於0.995，且方法偵測極限值為0.17~0.32 ng/g.。在本研究中利用超臨界流體萃取，聯結具有高選擇性的測定方法即選擇離子偵測(selected reaction monitoring, SRM)，可達到高的回收率以及好的精密度，而不需再進一步的淨化步驟。以本方法檢測從超市及藥局隨機所購買的大棗樣品，8個樣品中只有一個測定出有微量的黃麴毒素。This dissertation consists of three parts, which belong to the quality control of traditional Chinese medicines (TCMs). In this study, a liquid chromatography mass spectrometry (LC/MS) or liquid chromatography tandem mass spectrometry (LC/MS/MS) was developed to analyze TCMs, including Isatis indigotica (Banlangen & Daqingye) and Zizyphi jujube (Zizyphus fructus). The abstracts are as follows: In first part, a rapid, selective, and sensitive LC/APCI-MS method is developed in this study for detecting and analyzing tryptanthrin, indigo, and indirubin in Daqingye and Banlangen, which are, respectively, the leaves and roots of Isatis indigotica and Strobilanthes cusia in traditional Chinese medicine. The detection of the three active components is linear in concentrations ranging from 100 to 1500 ng/mL, the squared correlation coefficient is higher than 0.996, the precision as measured by the relative standard deviation is no larger than 9.5%, and the recovery is greater than 86.6%. The analysis of the 21 Banlangen samples led to considerably different conclusions on the contents of tryptanthrin, indigo, and indirubin in fresh leaves versus those in dried leaves. These results should shed some light on future plant selection and breeding. Compared with the traditional TLC and HPLC-UV methods, the new LC/APCI-MS approach has proven to be an optimal tool for detecting and analyzing the three marker compounds in the Chinese herbal medicines of Daqingye and Banlangen. In second part, the analytical method in this part was the same as part 1, which was also used to analyze three marker componets in Banlangen by LC/APCI-MS. However, in this part, the content difference in nanolized or un-nanolized Banlangen was compared. The results indicates that the contents of marker components in nanolized Banlangen (300nm) > in microlized Banlangen (5 m) > in Banlangen ground by lab (~0.5 mm). The content of tryptanthrin was increased as 2.4 folds, indigo 3.4 folds, and indirubin 3.3 folds comparing with/without nanolization. This phenomenon may be caused by the increasement of total surface area in nanolized Banlangen, then, increased the extraction efficiency by MeOH For the detection of concentrated extracts with herb, the method was failed to analyze the marker components in Banlangen due to the influence of complicate matrix. The complicate matrix was probably produced by formalization agent, and it could be moved with pretreament techniques, such as solid phase extraction (SPE). In third part, an integrated method supercritical fluid extraction (SFE) with LC/APCI-MS/MS was developed and successfully applied to quantify aflatoxins (AFs) in Zizyphi Fructus (fruits of Zizyphus jujube), a traditional Chinese medicine. To minimize the potential interferences caused by the complex matrix in Zizyphi Fructus, a SFE pretreatment was performed. In addition, electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) spectra were also compared. The results showed that the calibration curves of AFB1, AFB2, AFG1, and AFG2 were all linear over the range of concentration from 1 to 50 ng/g, the squared correlation coefficients (r2) were over 0.995, and the detection limits of method were between 0.17 and 0.32 ng/g. It showed high recovery and good precision in quantitating AFs in Zizyphi Fructus without further clean-up. Further, the fragmentation pathways of protonated AFs in APCI MS/MS were clearly proposed which could predict the existence of AFB or AFG series. To test the empirical validity of the proposed methodology in this paper, eight random samples of Zizyphi Fructus collected from supermarkets and traditional Chinese medicine stores in different geographical areas of Taiwan were analyzed. The results indicated that low levels of AFs were detected in only one of them.中文摘要……………………………………………….…………I 英文摘要…………………………………………………………IV 目次………………………………………………………….…VII 圖次………………………………………………….…………XII 表次…………………………………………………………….XIV 序論…………………………………………………….…………1 壹、LC/APCI-MS定量中藥材大青葉與板藍根之指標成份色胺酮、靛藍、及靛紅 一、前言 1.1 植物簡介………………………………………………………………4 1.2 研究動機………………………………………………..5 1.3 藥理活性………………………………………………..5 1.4 文獻上所報導之分析方法……………………….…….8 1.5 研究目的………………………………………………..8 二、實驗與分析方法 2.1 標準品及溶劑…………………….………..………….9 2.2 植物材料…………………………...........................10 2.3 標準品溶液及校正曲線的配製……………….………10 2.4 植物樣品的製備……………………………………….11 2.5 液相層析儀及液相層析條件…………………….……11 2.6 最佳化之質譜條件…………………………………….14 三、結果與討論 3.1 HPLC-UV 分析法……………………………………….15 3.2 質譜分析離子的選擇………………………………...17 3.3 修飾劑對離子化效率的影響……………………………19 3.4 選擇離子掃瞄偵測的質譜層析圖………………………21 3.5 線性範圍及偵測極限……………………………………21 3.6 精密度及回收率…………………………………………23 3.7 真實樣品的應用………………………………………..23 四、結論 結論……………………………………………………………28 貳、微奈米化板藍根之效能評估 一、前言 1.1 奈米技術…………………………….………………...29 1.2 中草藥之奈米化…………………………………………29 1.3 研究目的…………………………………………………30 二、實驗與分析方法 2.1分析方法………………………………………………….30 2.2 植物材料…………………………………………………31 2.3 奈米板藍根分析樣品的製備…………………………….31 三、結果與討論 3.1 HPLC-UV與 LC/MS分析法之比較…………………………34 3.2 板藍根微奈米化前後指標成份含量變化…………..….37 3.3 板藍根科學中藥奈米化前後之指標成份測定…………40 四、結論 結論……………………………………………………………42 參、超臨界二氧化碳萃取及利用LC/APCI-MS/MS定量分析大棗中的黃麴毒素含量 一、前言 1.1 黃麴毒素的簡介………………………………………….43 1.2 黃麴毒素的管制標準……………………………………45 1.3 大棗植物的簡介………………………………………….45 1.4 文獻上所報導之分析方法……………………………….46 1.5 研究目的…………………………………………………47 二、實驗與分析方法 2.1 試劑和植物材料………………………………………….47 2.2 SFE的萃取條件及樣品的製備……………………………48 2.3 液相層析條件…………………………………………..49 2.4 最佳化的質譜條件……………………………………….49 2.5 校正曲線及回收率……………………………………….51 三、結果與討論 3.1 SFE的收集溶劑及吸附材料………….………………….52 3.2 動相中修飾劑對質譜離子化效率的影響……………… 55 3.3 ESI及APCI方法的比較…………………………………….57 3.4 AFs特徵性的斷裂碎片………………..……………………59 3.5 校正曲線及偵測極限……………………………………….63 3.6 回收率及再現性…………………………………………….65 3.7 真實樣品的分析…………………………………………….67 四、結論 結論…………………………………….…………………………67 參考文獻…………………………..……………………………69 附錄 附錄一、中英文名稱對照表……………………….………. 附錄1 附錄二、板藍根中已知化學成分一覽表……………..…. 附錄6 附錄三、電灑游離法及大氣壓化學游離法的原理與參數……附錄11 附錄四、離子阱質量分析器之介紹……………….…………. 附錄19 附錄五、第一部份之期刊發表…………………………………附錄23 附錄六、第二部份之期刊發表…………………………………附錄29 附錄七、SFE原理及最佳化之萃取參數…………….…………附錄35 附錄八、第三部份之期刊發表…………………………………附錄4

Quality Control of Traditional Chinese Medicine: Analysis of marker components in Banlangen and aflatoxin in Zizyphus fructus by LC/MS

本論文共分為三部分，皆屬中藥材品管之研究，研究之中藥材包括板藍根、大青葉及大棗，所用之分析儀器為液相層析質譜儀(LC/MS)及液相層析串聯質譜儀(LC/MS/MS)，其摘要內容如下: 第一部份主要是利用液相層析質譜儀之大氣壓化學游離法(LC/APCI-MS) 正離子模式來偵測中藥材大青葉(Daqingye)及板藍根(Isatis indigotica, Banlangen)中三個指標成份色胺酮(tryptanthrin)、靛藍(indigo)、靛紅(indirubin)的含量；在本篇研究中，成功地發展出一種快速、具選擇性、及高靈敏性的LC/APCI-MS的方法，用以偵測及分析大青葉及板藍根中的色胺酮、靛藍、及靛紅之含量，此三個指標成份的偵測範圍為100~1500 ng/mL，線性相關係數的平方值大於0.996，精密度的相對標準偏差值(RSD)小於9.5%，且回收率大於86.6%。將此方法應用於真實植物樣品的分析，選擇21個來自不同地區的板藍根樣品，分析其新鮮、乾燥葉、及乾燥根中色胺酮、靛藍、及靛紅的含量，其結果可做為此植物在選種及育種方面的參考。最後，比較液相層析紫外光偵測器(HPLC-UV)的方法，此LC/APCI-MS的方法可以成功地用來分析大青葉及板藍根中的三種指標成份的含量。 第二部份是以液相層析質譜儀之大氣壓化學游離法正離子模式，來偵測中藥材板藍根中之指標成份色胺酮、靛藍、靛紅的含量，所不同的地方在於本部份著眼於分析比較板藍根奈米化顆粒前後，指標成份含量的差異性，其結果為奈米化板藍根藥材(粒徑300 nm) 之三種分析物含量 > 微米化板藍根(粒徑5 µm) > 實驗室的研磨尺寸(~0.5 mm)，其中色胺酮之含量在微奈米化後約增為原來2.4倍，靛藍含量增加3.4倍以上，而靛紅則增加3.3倍，這說明了板藍根藥材在奈米化後，可能由於其總表面積變大，增加了甲醇在單位時間的萃出效果。 對於板藍根科學中藥的測定，因受到大量的基質干擾而無法準確的定量分析，此基質干擾有可能是來自科學中藥中的定型劑，須先了解定型劑的種類為何，再配合前處理步驟，如固相萃取，用以淨化其複雜基質。 第三部分主要是探討利用液相層析串聯質譜儀之大氣壓化學游離法(LC/APCI-MS/MS)正離子模式來對中藥材大棗(Zizyphi jujube, Ziphus fructus)之黃麴毒素作定量分析。本部份以超臨界流體萃取(supercritical fluid extraction, SFE)技術配合LC/APCI-MS/MS的分析方法，可成功的應用在傳統中藥大棗中黃麴毒素B1，B2，G1，G2 (AFB1，B2，G1，G2)的測定。為了減少大棗中複雜基質的干擾，須利用超臨界流體萃取技術的高選擇性特點配合LC/MS/MS測定，檢測奈克級的黃麴毒素。游離化的模式包含電灑游離法(electrospray ionization, ESI)以及大氣壓化學游離法(atmosphere pressure chemical ionization, APCI)，並比較二者間的偵測效率，以達到最佳化的質譜測定條件。AFB1，B2，G1，G2的校正曲線濃度在介於1 ~ 50 ng/g 時呈線性，其線性相關係數的平方值(r2)大於0.995，且方法偵測極限值為0.17~0.32 ng/g.。在本研究中利用超臨界流體萃取，聯結具有高選擇性的測定方法即選擇離子偵測(selected reaction monitoring, SRM)，可達到高的回收率以及好的精密度，而不需再進一步的淨化步驟。以本方法檢測從超市及藥局隨機所購買的大棗樣品，8個樣品中只有一個測定出有微量的黃麴毒素。This dissertation consists of three parts, which belong to the quality control of traditional Chinese medicines (TCMs). In this study, a liquid chromatography mass spectrometry (LC/MS) or liquid chromatography tandem mass spectrometry (LC/MS/MS) was developed to analyze TCMs, including Isatis indigotica (Banlangen & Daqingye) and Zizyphi jujube (Zizyphus fructus). The abstracts are as follows: In first part, a rapid, selective, and sensitive LC/APCI-MS method is developed in this study for detecting and analyzing tryptanthrin, indigo, and indirubin in Daqingye and Banlangen, which are, respectively, the leaves and roots of Isatis indigotica and Strobilanthes cusia in traditional Chinese medicine. The detection of the three active components is linear in concentrations ranging from 100 to 1500 ng/mL, the squared correlation coefficient is higher than 0.996, the precision as measured by the relative standard deviation is no larger than 9.5%, and the recovery is greater than 86.6%. The analysis of the 21 Banlangen samples led to considerably different conclusions on the contents of tryptanthrin, indigo, and indirubin in fresh leaves versus those in dried leaves. These results should shed some light on future plant selection and breeding. Compared with the traditional TLC and HPLC-UV methods, the new LC/APCI-MS approach has proven to be an optimal tool for detecting and analyzing the three marker compounds in the Chinese herbal medicines of Daqingye and Banlangen. In second part, the analytical method in this part was the same as part 1, which was also used to analyze three marker componets in Banlangen by LC/APCI-MS. However, in this part, the content difference in nanolized or un-nanolized Banlangen was compared. The results indicates that the contents of marker components in nanolized Banlangen (300nm) > in microlized Banlangen (5 µm) > in Banlangen ground by lab (~0.5 mm). The content of tryptanthrin was increased as 2.4 folds, indigo 3.4 folds, and indirubin 3.3 folds comparing with/without nanolization. This phenomenon may be caused by the increasement of total surface area in nanolized Banlangen, then, increased the extraction efficiency by MeOH For the detection of concentrated extracts with herb, the method was failed to analyze the marker components in Banlangen due to the influence of complicate matrix. The complicate matrix was probably produced by formalization agent, and it could be moved with pretreament techniques, such as solid phase extraction (SPE). In third part, an integrated method supercritical fluid extraction (SFE) with LC/APCI-MS/MS was developed and successfully applied to quantify aflatoxins (AFs) in Zizyphi Fructus (fruits of Zizyphus jujube), a traditional Chinese medicine. To minimize the potential interferences caused by the complex matrix in Zizyphi Fructus, a SFE pretreatment was performed. In addition, electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) spectra were also compared. The results showed that the calibration curves of AFB1, AFB2, AFG1, and AFG2 were all linear over the range of concentration from 1 to 50 ng/g, the squared correlation coefficients (r2) were over 0.995, and the detection limits of method were between 0.17 and 0.32 ng/g. It showed high recovery and good precision in quantitating AFs in Zizyphi Fructus without further clean-up. Further, the fragmentation pathways of protonated AFs in APCI MS/MS were clearly proposed which could predict the existence of AFB or AFG series. To test the empirical validity of the proposed methodology in this paper, eight random samples of Zizyphi Fructus collected from supermarkets and traditional Chinese medicine stores in different geographical areas of Taiwan were analyzed. The results indicated that low levels of AFs were detected in only one of them.中文摘要……………………………………………….…………I 英文摘要…………………………………………………………IV 目次………………………………………………………….…VII 圖次………………………………………………….…………XII 表次…………………………………………………………….XIV 序論…………………………………………………….…………1 壹、LC/APCI-MS定量中藥材大青葉與板藍根之指標成份色胺酮、靛藍、及靛紅 一、前言 1.1 植物簡介………………………………………………………………4 1.2 研究動機………………………………………………..5 1.3 藥理活性………………………………………………..5 1.4 文獻上所報導之分析方法……………………….…….8 1.5 研究目的………………………………………………..8 二、實驗與分析方法 2.1 標準品及溶劑…………………….………..………….9 2.2 植物材料…………………………...........................10 2.3 標準品溶液及校正曲線的配製……………….………10 2.4 植物樣品的製備……………………………………….11 2.5 液相層析儀及液相層析條件…………………….……11 2.6 最佳化之質譜條件…………………………………….14 三、結果與討論 3.1 HPLC-UV 分析法……………………………………….15 3.2 質譜分析離子的選擇………………………………...17 3.3 修飾劑對離子化效率的影響……………………………19 3.4 選擇離子掃瞄偵測的質譜層析圖………………………21 3.5 線性範圍及偵測極限……………………………………21 3.6 精密度及回收率…………………………………………23 3.7 真實樣品的應用………………………………………..23 四、結論 結論……………………………………………………………28 貳、微奈米化板藍根之效能評估 一、前言 1.1 奈米技術…………………………….………………...29 1.2 中草藥之奈米化…………………………………………29 1.3 研究目的…………………………………………………30 二、實驗與分析方法 2.1分析方法………………………………………………….30 2.2 植物材料…………………………………………………31 2.3 奈米板藍根分析樣品的製備…………………………….31 三、結果與討論 3.1 HPLC-UV與 LC/MS分析法之比較…………………………34 3.2 板藍根微奈米化前後指標成份含量變化…………..….37 3.3 板藍根科學中藥奈米化前後之指標成份測定…………40 四、結論 結論……………………………………………………………42 參、超臨界二氧化碳萃取及利用LC/APCI-MS/MS定量分析大棗中的黃麴毒素含量 一、前言 1.1 黃麴毒素的簡介………………………………………….43 1.2 黃麴毒素的管制標準……………………………………45 1.3 大棗植物的簡介………………………………………….45 1.4 文獻上所報導之分析方法……………………………….46 1.5 研究目的…………………………………………………47 二、實驗與分析方法 2.1 試劑和植物材料………………………………………….47 2.2 SFE的萃取條件及樣品的製備……………………………48 2.3 液相層析條件…………………………………………..49 2.4 最佳化的質譜條件……………………………………….49 2.5 校正曲線及回收率……………………………………….51 三、結果與討論 3.1 SFE的收集溶劑及吸附材料………….………………….52 3.2 動相中修飾劑對質譜離子化效率的影響……………… 55 3.3 ESI及APCI方法的比較…………………………………….57 3.4 AFs特徵性的斷裂碎片………………..……………………59 3.5 校正曲線及偵測極限……………………………………….63 3.6 回收率及再現性…………………………………………….65 3.7 真實樣品的分析…………………………………………….67 四、結論 結論…………………………………….…………………………67 參考文獻…………………………..……………………………69 附錄 附錄一、中英文名稱對照表……………………….………. 附錄1 附錄二、板藍根中已知化學成分一覽表……………..…. 附錄6 附錄三、電灑游離法及大氣壓化學游離法的原理與參數……附錄11 附錄四、離子阱質量分析器之介紹……………….…………. 附錄19 附錄五、第一部份之期刊發表…………………………………附錄23 附錄六、第二部份之期刊發表…………………………………附錄29 附錄七、SFE原理及最佳化之萃取參數…………….…………附錄35 附錄八、第三部份之期刊發表…………………………………附錄4

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一種具有生物活性的玉米黃素棕櫚酸酯的結晶純化方法，尤特指利用超音波攪拌溶劑，從除醣的枸杞乾糙果粒中提取玉米黃素棕櫚酸酯，接著以管柱層析純化程序產製富含玉米黃素棕櫚酸酯分劃層；最後，再以液－液抗溶結晶純化程序，得到純品98.3%的玉米黃素棕櫚酸酯，為其特徵者

Separation of sight-protecting zeaxanthin from Nannochloropsis oculata by using supercritical fluids extraction coupled with elution chromatography

Microalgae is a potent source of carotenoids and is regarded as functional foods consumed by people worldwide. In this study, supercritical fluid extraction coupled with elution chromatography was employed to recover six carotenoids including zeaxanthin from Nannochloropsis oculata and extraction efficiency of zeaxanthin was determined. The amount of the zeaxanthin (400 mg/g) in the elution fraction increased more than twenty folds from that of the SFE extract (13.17 mg/g). The extracts were subsequently subjected to tests of antioxidant capacities in 2,2-diphenyl-2-picrylhydrazyl hydrate (DPPH) assay, 2-2'-azino-bis (3-ethylbenzo- thiazoline-6-sulfonic acid (ABTS)) assay and adult retinal pigment epithelium-19 (ARPE-19) cells. Experimental results demonstrate that other components than zeaxanthin may play a crucial role in the anti-oxidative ability of the extracts from N. oculata. This work provides a useful method to recover bioactive anti-oxidative zeaxanthin from microalgae of N. oculata by using modified supercritical fluids extraction

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Clinical Outcomes of Patients with Resected Oral Cavity Cancer and Simultaneous Second Primary Malignancies

<div><p>Objectives</p><p>Simultaneous second primary tumors (SSPT) are not uncommon in patients with oral cavity squamous cell carcinoma (OSCC) living in areas where the habit of betel quid chewing is widespread. We sought to identify the main prognostic factors in OSCC patients with SSPT and incorporate them into a risk stratification scheme.</p><p>Methods</p><p>A total of 1822 consecutive patients with primary OSCC treated between January 1996 and February 2014 were analyzed for the presence of SSPT. The 18-month and 5-year overall survival (OS) rates served as the main outcome measures.</p><p>Results</p><p>Of the 1822 patients, 77 (4%) were found to have SSPT (i.e, two malignancies identified within one month of each other). The 18-month and 5-year OS rates in patients without SSPT and with SSPT were 82% and 69%, and 72% and 53%, respectively (<i>p</i> = 0.0063). Patients with SSPT were further divided into patients with either esophageal cancer or hepatocellular carcinoma (eso-HCC subgroup, n = 8) and other tumors (NO eso-HCC subgroup, n = 69). After multivariate analysis, neck nodal extracapsular spread (ECS, n = 18) and the presence of eso-HCC were identified as independent adverse prognostic factors. The 18-month OS rates of SSPT patients with both eso-HCC and ECS (n = 5) <i>vs</i>. the remaining patients (n = 72) were 0% and 78%, respectively (<i>p</i> < 0.0001).</p><p>Conclusion</p><p>OSCC patients with neck nodal ECS and esophageal cancer or hepatocellular carcinoma as SSPT have a dismal short-term prognosis.</p></div